In vitro production of medial ganglionic eminence precursor cells

ABSTRACT

Methods and systems for generating MGE precursor cells in vitro as well as compositions of enriched MGE precursor cells are provided. The methods and systems provide efficient production of MGE precursors. The methods and systems disclosed herein provide functional MGE precursors which differentiate into functional GABAergic interneurons.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/783,594 filed Mar. 14, 2013, which application isincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. RO1MH081880 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INTRODUCTION

Inhibitory interneurons account for about 20% of neurons in the cerebralcortex. Deficiencies of interneurons are implicated in severalneurological disorders. Most cortical interneurons originate in themedial ganglionic eminence (MGE) of the developing ventral telencephalonregion of the brain.

Mouse MGE transplants were shown to ameliorate multiple rodent models ofneurological disorders, suggesting human MGE cells may represent aunique therapeutic candidate.

However, in vitro methods for efficient generation of cells havingcharacteristics of cells of the MGE are not available.

As such, there is a need for method for efficiently generating MGEprecursor cells in vitro and for cell populations enriched in MGEprecursor cells.

SUMMARY

Methods and systems for generating MGE precursor cells in vitro as wellas compositions of enriched MGE precursor cells are provided. Themethods and systems provide efficient production of functional MGEprecursors, which differentiate into functional GABAergic interneurons.

A method of producing medial ganglionic eminence (MGE) precursor cellsfrom primate pluripotent stem (pPS) cells is provided.

In certain embodiments, the method includes culturing the pPS cells in aserum free medium containing an activator of sonic hedgehog pathway anda neural inducing supplement to generate the MGE precursor cells. ThepPS cell may be cultured in an adherent culture or in a suspensionculture.

In certain embodiments, the method includes culturing the pPS cells in aserum free medium containing an activator of sonic hedgehog pathway anda neural inducing supplement to generate embryoid bodies (EBs), whereinthe EBs comprise the MGE precursor cells.

In certain cases, the neural inducing supplement may be B27. In certaincases, the neural inducing supplement may be NS21.

In certain embodiments, the pPS cells may be human pluripotent stem(hPS) cells. The hPS cells may be human embryonic stem (hES) cells orinduced pluripotent stem (iPS) cells.

In certain embodiments, the pPS cells may be induced to differentiateprior to culturing the pPS cells in the serum free medium comprising theactivator of sonic hedgehog pathway and the neural inducing supplement.For example, the pPS cells may be induced to differentiate by overgrowthof the pPS cell culture, or by culturing pPS cells in suspension inculture vessels having a substrate with low adhesion, culturing pPS inabsence of feeder layer, or adding a differentiation factor such as FGFbefore culturing the pPS cells in the serum free medium comprising theactivator of sonic hedgehog pathway and the neural inducing supplement.

In certain embodiments, the method may include isolating the EBs;plating the isolated EBs on an adherent substrate to provide adherentEBs; and culturing the adherent EBs.

In certain embodiments, the method may include isolating the EBs;dissociating the EBs mechanically or enzymatically to produce singlecells or clusters of cells; plating the dissociated cells on an adherentsubstrate to provide an adherent monolayer; and culturing the adherentmonolayer.

In certain embodiments, the method may include isolating the EBs;dissociating the EBs mechanically or enzymatically to produce singlecells or dusters of cells; plating the dissociated cells on a cellularfeeder layer to provide an adherent co-culture; and culturing theadherent co-culture.

In certain embodiments, the method may include isolating the EBs,adherent EBs, monolayer, or co-cultures; dissociating the EBs, adherentEBs, monolayer, or co-cultures mechanically or enzymatically to producesingle cells; incubating the single cells with an antibody to a cellsurface marker for MGE precursor cells; and isolating the precursorcells.

In certain embodiments, the method may include isolating the EBs,adherent EBs, monolayer, co-cultures, dissociated cultures, or isolatedprecursor cells; and adding a cryoprotectant, such as, antifreezecompounds, e.g., glycols (glycerol, ethylene glycol, propylene glycol),dimethyl sulfoxide (DMSO), or sucrose.

In certain cases, a method of producing medial ganglionic eminence (MGE)precursor cells from primate pluripotent stem (pPS) cells may includeculturing the pPS cells in a serum free medium to generate embryoidbodies (EBs), wherein the EBs include the MGE precursor cells, whereinthe serum free medium includes an activator of sonic hedgehog pathway,an inhibitor of Rho-associated kinase (ROCK), an inhibitor of SMAD, aninhibitor of Wnt and B27.

The pPS cells are human pluripotent stem (hPS) cells may be humanembryonic stem (hES) cells or induced pluripotent stem (iPS) cells.

In certain cases, the method may further include isolating the EBs;plating the isolated EBs on an adherent substrate to provide adherentEBs; and culturing the adherent EBs.

In certain embodiments, the adherent EBs are cultured in a serum freemedium comprising an activator of sonic hedgehog pathway, an inhibitorof SMAD, an inhibitor of Wnt, and B27.

In certain embodiments, the adherent EBs are cultured in a serum freemedium that does not contain an inhibitor of ROCK.

A method for producing inhibitory interneurons is provided, the methodmay include isolating the EBs, adherent EBs, monolayer, co-cultures,dissociated cultures, or sorted cells produced as described above;producing a cell suspension of the isolated cells and transplanting cellsuspensions into the primate nervous system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (panels A-B) illustrate the generation of MGE-like PrecursorCells.

FIG. 2 (Panels A-F) illustrate hESC-MGE-like progenitors exhibit VZ andSVZ Radial Glial Stern Cell-like Divisions.

FIG. 3 (Panels A-F) illustrate hESC-MGE-like progenitors differentiateinto neurons with properties of telencephalic GABAergic interneurons.

FIG. 4 (Panels A-H) depict microarray gene expression profiling ofhESC-MGE-like NKX2.1-GFP+ Cell Populations,

FIG. 5 (Panels A-J) illustrate hESC-MGE-like cell-derived GABAergicinterneuron maturation and firing properties.

FIG. 6 (Panels A-J) illustrate GABAergic Synaptic Properties ofhESC-derived Interneurons.

FIG. 7 (Panels A-H) show hESC-derived MGE-like interneuron precursorcell maturation and functional integration in the mouse brain.

FIG. 8 (Panels A-F) provide a schematic of differentiation protocols andFACS analysis of differentiated hESCs.

FIG. 9 illustrates hESC-derived cells have telencephalic MGE-likeidentity and GABAergic neuronal fate.

FIG. 10 (Panels A-F) depict transcript expression profiling ofhESC-derived NKX2.1-GFP+ cells.

FIG. 11 depicts maturation of hESC-derived MGE-like cells into GABAergicinterneuron subtypes.

FIG. 12 (Panels A-C) show development of interneuron subtypes in humanfetal cortex and MGE, and in cultures derived from human fetal MGE.

FIG. 13 (Panels A-F) show maturation of hESC-derived interneuron firingproperties.

FIG. 14 (Panels A-G) depict maturation of hESC-derived MGE-likeinterneurons and subtype firing properties in the mouse brain.

FIG. 15 provides a summary of marker expression during differentiationfrom hESCs.

FIG. 16 provides a summary of hESC differentiation protocoloptimization, animal transplantation, and tumor incidence.

FIG. 17 depicts MGE precursor cells differentiated in vitro from hESCline ESI17.

FIG. 18 depicts MGE precursor cells differentiated in vitro from hESCline ESI35.

FIG. 19 depicts MGE precursor cells differentiated in vitro from hESCline ESI51.

FIG. 20 depicts MGE precursor cells differentiated in vitro from hESCline H9.

FIG. 21 illustrates generation of MGE precursor cells by differentiationof naïve human pluripotent stem cells.

FIG. 22 (Panels A-N) illustrates utilization of an MGE-enriched enhancersequence for the selection and purification of interneurons derived fromMGE precursor cells generated by differentiation of hPSC.

FIG. 23 (Rows A-D) depict generation of MGE derived interneurons usinglong-term suspension culture.

FIG. 24 (Panels A-E) illustrates that numerous small molecule inhibitorsof BMP and WNT signaling pathways are effective in inducingdifferentiation of hESCs into MGE precursor cells.

DEFINITIONS

As used herein, “embryoid body”, “embryoid bodies”, “EBs” or “EB cells”typically refers to a morphological, three-dimensional, or organoid-typestructure comprised of a population of undifferentiated anddifferentiated cells which are derived from pluripotent stem cells(e.g., primate pluripotent stem cells (pPS), embryonic stem (ES) cells,induced pluripotent stem (iPS) cells) that have undergonedifferentiation, Under culture conditions suitable for EB formation, EScells proliferate and form small mass of cells that begin todifferentiate. In the first phase of differentiation, usuallycorresponding, to about days 1-4 of differentiation for human cells, thesmall mass of cells forms a layer of endodermal cells on the outerlayer, and is considered a “simple embryoid body.” In the second phase,usually corresponding to about days 3-20 post-differentiation for humancells, “complex embryoid bodies” are formed, which are characterized byextensive differentiation of ectodermal and mesodermal cells andderivative tissues. As used herein, the term “embryoid bodies” or “EB”encompasses both simple and complex embryoid bodies unless otherwiserequired by context. The determination of when embryoid bodies haveformed in a culture of ES/iPS cells is routinely made by persons ofskill in the art by, for example, visual inspection of the morphology,detection of cell markers. Floating masses of about 20 cells or more(e.g., ES/iPS cells) are considered to be suspension embryoid bodies(sEB). (see. e.g., Schmitt, R., et al. (1991) Genes Dev. 5:728-740;Doetschman, T. C., et al. (1985) J. Embryol. Exp. Morph. 87:27-45).Suspension EBs can be plated onto an adherent substrate to generateadherent EBs (aEB).

As used herein, “medial ganglionic eminence (MGE) precursor cell(s)” or“MGE neural precursor cells,” refer to a population of mitotic andpost-mitotic cells that express the markers expressed by cells in theMGE region of the developing brain. In general MGE precursor cellsexpress markers such as, homeobox gene Nkx2.1, LIM-homeobox genes Lhx6,Lhx7, or Lhx8. MGE precursor cells are capable of differentiating intointerneurons under suitable differentiation conditions.

By “pluripotent stem cell” or “pluripotent cell” it is meant a cell thathas the ability under appropriate conditions of producing progeny ofseveral different cell types that are derivatives of all of the threegerminal layers (endoderm, mesoderm, and ectoderm). Pluripotent stemcells are capable of forming teratomas. Examples of pluripotent stemcells are embryonic stem (ES) cells, embryonic germ stem (EG) cells,embryonal carcinoma stem (EC) cells, and induced pluripotent stem (iPS)cells. PS cells may be from any organism of interest, including, e.g.,human; primate; non-human primate; canine; feline; neurine; equine;porcine; avian; camel; bovine; ovine, and so on.

By “embryonic stem cell” or “ES cell” it is meant a cell that a) canself-renew, b) can differentiate to produce all types of cells in anorganism, and c) is derived from a developing organism or is anestablished ES cell line which was derived from a developing organism.ES cell may be derived from the inner cell mass of the blastula, or fromthe epiblast, of a developing organism. ES cell may be derived from ablastomere generated by single blastomere biopsy (SBB) involving removalof a single blastomere from the developing organism. In general, SBBprovides a non-destructive alternative to inner cell mass isolation. SBBand generation of hES cells from the biopsied blastomere is described inCell Stem Cell, 2008 Feb. 7; 2(2):113-7. ES cells can be cultured over along period of time while maintaining the ability to differentiate intoall types of cells in an organism. In culture, ES cells typically growas flat colonies with large nucleo-cytoplasmic ratios, defined bordersand prominent nucleoli. In addition, hES cells express SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examplesof methods of generating and characterizing ES cells may be found in,for example, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, thedisclosures of which are incorporated herein by reference. Examples ofES cells include nave ES cells.

By “embryonic germ stem cell”, embryonic germ cell” or “EG cell” it ismeant a cell that a) can self-renew, b) can differentiate to produce alltypes of cells in an organism, and c) is derived from germ cells andgerm cell progenitors, e.g. primordial germ cells, i.e. those that wouldbecome sperm and eggs. Embryonic germ cells (EG cells) are thought tohave properties similar to embryonic stem cells as described above.Examples of methods of generating and characterizing EG cells may befound in, for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al.,(1992) Cell 70:841; Shamblott, M., et al, (2001) Proc. Natl. Acad. Sci.USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA,95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, thedisclosures of which are incorporated herein by reference.

By “induced pluripotent stem cell” or “iPS cell” it is meant a cell thata) can self-renew, b) can differentiate to produce all types of cells inan organism, and c) is derived from a somatic cell. iPS cells have an EScell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nucleoli. Inaddition, iPS cells express one or more key pluripotency markers knownby one of ordinary skill in the art, including but not limited toAlkaline Phosphatase, SSEA3, SSEA4, Sox 2, Oct3/4, Nanog, TRA160,TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. iPS cellsmay be generated by providing the cell with “reprogramming factors”,i.e., one or more, e.g., a cocktail, of biologically active factors thatact on a cell to alter transcription, thereby reprogramming a cell topluripotency. Examples of methods of generating and characterizing iPScells may be found in, for example, Application Nos. US20090047263,US20090068742, US20090191159, US20090227032, US20090246875, andUS20090304646, the disclosures of which are incorporated herein byreference.

By “somatic cell” it is meant any cell in an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells arecells that have differentiated sufficiently that they will not naturallygenerate cells of all three germ layers of the body, i.e., ectoderm,mesoderm and endoderm. For example, somatic cells would include bothneurons and neural progenitors, the latter of which may be able toself-renew and naturally give rise to all or some cell types of thecentral nervous system but cannot give rise to cells of the mesoderm orendoderm lineages.

The term “cell line” refers to a population of largely or substantiallyidentical cells that has typically been derived from a single ancestorcell or from a defined and/or substantially identical population ofancestor cells. The cell line may have been or may be capable of beingmaintained in culture for an extended period (e.g., months, years, foran unlimited period of time).

By “endoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to the gastrointestinal tract, respiratorytract, endocrine glands and organs, certain structures of the auditorysystem, and certain structures of the urinary system.

By “mesoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to muscles, cartilage, bones, dermis, thereproductive system, adipose tissue, connective tissues of the gut,peritoneum, certain structures of the urinary system, mesothelium,notochord, and spleen.

By “ectoderm” it is meant the germ layer formed during animalembryogenesis that gives rise to the nervous system, tooth enamel,epidermis, hair, nails, and linings of mucosal tissues.

By “bone morphogenic proteins” or “BMPs” it is meant the family ofgrowth factors that is a subfamily of the transforming growth factor β(TGF β) superfamily. BMPs (e.g. BMP1, BMP2, BMP3, BMP4, BMP5, BMP6,BMP7, BMP8a, BMP8b, BMP9/GDF, BMP10, BMP11/GDF11, BMP12/GDF7,BMP13/GDF6, BMP14/GDF5, BMP15/GDF9B) were first discovered by theirability to induce the formation of bone and cartilage. BMPs interactwith specific receptors on the cell surface, referred to as bonemorphogenetic protein receptors (BMPRs). Signal transduction throughBMPRs results in mobilization of members of the SMAD family of proteins,which in turn modulate transcription of target genes. Inhibitors of BMPsignaling, can readily be identified by one of ordinary skill in the artby any of a number of methods, for example competitive binding assaysfor binding to BMP or BMP receptors, functional assays, e.g., measuringenhancement of activity of downstream signaling proteins such asrelocalisation of SMADs, such as, BR-Smad to the nucleus andtranscriptional activation of downstream gene targets as known in theart.

By “transforming growth factor betas”, “TGF-βs”, and “TGFBs” it is meantthe TGFB secreted proteins belonging to the subfamily of thetransforming growth factor β (TGFβ) superfamily. TGFBs (TGFB1, TGFB2,TGFB3) are multifunctional peptides that regulate proliferation,differentiation, adhesion, and migration and in many cell types. Themature peptides may be found as homodimers or as heterodimers with otherTGFB family members. TGFBs interact with transforming growth factor betareceptors (TGF-βRs, or TGFBRs) on the cell surface, which bindingactivates MAP kinase-, Akt-, Rho- and Rac/cdc42-directed signaltransduction pathways, the reorganization of the cellular architectureand nuclear localization of SMAD proteins, and the modulation of targetgene transcription. Inhibitors of TGFB signaling, can be readily beidentified by one of ordinary skill in the art by any of a number ofmethods, for example competitive binding assays for binding to TGFB orTGFB receptors, or functional assays, e.g. measuring suppression ofactivity of downstream signaling proteins such as MAPK, Akt, Rho, Rac,and SMADs, e.g., AR-Smad, etc., as well known in the art.

By “Wnts” it is meant the family of highly conserved secreted signalingmolecules which play key roles in both embryogenesis and mature tissues.The human Wnt gene family has at least 19 members (Wnt-1, Wnt-2,Wnt-2B/Wnt-13, Wnt-3, Wnt3a, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A,Wnt-7B, Wnt-8A, Writ-8B, Wnt-9A/Wnt-14, Wnt-9B/Wnt-15, Wnt-10A, Wnt-10B,Writ-11, Wnt-16). Wnt proteins modulate cell activity by binding to Wntreceptor complexes that include a polypeptide from the Frizzled (Fz)family of proteins and a polypeptide of the low-density lipoproteinreceptor (LDLR)-related protein (LRP) family of proteins. Once activatedby Wnt binding, the Wnt receptor complex will activate one or moreintracellular signaling cascades. These include the canonical Wntsignaling pathway; the Wnt/planar cell polarity (Wnt/PCP) pathway; andthe Wnt-calcium (Wnt/Ca2+) pathway.

By culturing under “non-adherent conditions” it is meant culturing underconditions that suppress the adhesion of cells to the vessel in whichthey are cultured, e.g., the bottom of a tissue culture plate or flask.In some instances, the cells are naturally non-adherent, i.e., they willnot adhere to a surface unless the surface is coated with a matrixcomposition, e.g., fibronectin, laminin, poly-ornithin, poly-lysine,collagen IV, matrigel, and polycarbonate membranes. In some instances,cells may be maintained in a non-adherent state by agitating theculture.

By culturing under “adherent conditions” it is meant culturing underconditions that promote the adhesion of cells to the container in whichthey are cultured, e.g. the bottom of a tissue culture plate or flask.In some instances, cells may be induced to adhere to the containersimply by keeping the culture stationary. In some instances, the wall ofthe container to which it is desirable to promote adhesion may be coatedwith a composition to which the cells may adhere, e.g., fibronectin,laminin, poly-ornithin, poly-lysine, collagen IV, matrigel, andpolycarbonate membranes.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving the disease, i.e., causing regression of the disease. Thetherapeutic agent may be administered before, during or after the onsetof disease or injury. The treatment of ongoing disease, where thetreatment stabilizes or reduces the undesirable clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to complete loss of function in the affected tissues.The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

The terms “individual”, “subject”, “host”, and “patient” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

The term “medium” in context of cell culture or the phrase “cell culturemedium” or “cell medium” refer to a cellular growth medium suitable forculturing of a cell population of interest. Examples of cell culturemedium include Minimum Essential Medium (MEM), Eagle's Medium,Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified. EagleMedium: Nutrient Mixture F-12 (DMEM/F12), F10 Nutrient Mixture, Ham'sF10 Nutrient Mix, Ham's F12 Nutrient Mixture, Medium 199, RPMI, RPMI1640, reduced serum medium, basal medium (BME), DMEM/F12 (1:1),Neurobasal medium, and the like, and combinations thereof. The medium orcell culture medium may be modified by adding one or more factors, suchas, supplements, differentiation factors, anti-apoptotic agents.

The term “isolated” in context of cells or cell population refers tocells that are in an environment other than their native environment,such as, apart from tissue of an organism.

The phrase “differentiation factor(s)” as used herein refers to theagent(s) that are included in the medium for culturing cells of thepresent disclosure, which agent(s) promote the differentiation of thecells from a first cell type to a second cell type, where the secondcell type is differentiated compared to the first cell type.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, pluripotent embryonic stem cells can differentiateto lineage-restricted precursor cells. These in turn can bedifferentiated further to cells further down the pathway, or to anend-stage differentiated cell, such as GABAergic interneuron.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. pPS cellpopulations are said to be “essentially free” of feeder cells if thecells have been grown through at least one round after splitting inwhich fresh feeder cells are not added to support the growth of pPScells.

As used herein, “expression” and grammatical equivalents thereof, in thecontext of a marker, refers to production of the marker as well as levelor amount of the marker. For example, expression of a marker or presenceof a marker in a cell or a cell is positive for a marker, refers toexpression of the marker at a level that is similar to a positivecontrol level. The positive control level may be determined by the levelof the marker expressed by a cell known to have the cell fate associatedwith the marker. Similarly, absence of expression of a marker or a cellis negative for a marker, refers to expression of the marker at a levelthat is similar to a negative control level. The negative control levelmay be determined by the level of the marker expressed by a cell knownto not have the cell fate associated with the marker. As such, absenceof a marker does not simply imply an undetectable level of expression ofthe marker, in certain cases, a cell may express the marker but theexpression may be low compared to a positive control or may be at alevel similar to that of a negative control.

As used herein, “marker” refers to any molecule that can be measured ordetected. For example, a marker can include, without limitations, anucleic acid, such as, a transcript of a gene, a polypeptide product ofa gene, a glycoprotein, a carbohydrate, a glycolipid, a lipid, alipoprotein, a carbohydrate, or a small molecule (for example, amolecule having a molecular weight of less than 10,000 amu).

A “variant” polypeptide means a biologically active polypeptide asdefined below having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%sequence identity with a native sequence polypeptide, Such variantsinclude polypeptides wherein one or more amino acid residues are addedat the N- or C-terminus of, or within, the native sequence; from aboutone to forty amino acid residues are deleted, and optionally substitutedby one or more amino acid residues; and derivatives of the abovepolypeptides, wherein an amino acid residue has been covalently modifiedso that the resulting product has a non-naturally occurring amino acid.Ordinarily, a biologically active variant will have an amino acidsequence having at least about 90% amino acid sequence identity with anative sequence polypeptide, at least about 95%, or at least about 99%.The variant polypeptides can be naturally or non-naturally glycosylated,i.e., the polypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringprotein. The variant polypeptides can have post-translationalmodifications not found on the natural polypeptide.

The terms ‘enriching” or “enriched” are used interchangeably herein andmean that the yield (fraction) of cells of one type is increased by atleast 10% over the fraction of cells of that type in the startingculture or preparation.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

DETAILED DESCRIPTION

As noted above, methods and systems for generating MGE precursor cellsin vitro as well as compositions of enriched MGE precursor cells areprovided. The methods and systems provide efficient production offunctional MGE precursors, which differentiate into functional GABAergicinterneurons.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aSMAD inhibitor” includes a plurality of such inhibitors and reference to“the ROCK inhibitor” includes reference to one or more ROCK inhibitorand equivalents thereof known to those skilled in the art, and so forth.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Method for Generating MGE Precursor Cells

In certain embodiments, a method of producing medial ganglionic eminence(MGE) precursor cells from primate pluripotent stem (pPS) cells isprovided.

In general, the pPS are maintained in an undifferentiated state till themethod for production of MGE precursor cells is commenced.

The method may include culturing the pPS cells in a serum free mediumcomprising an activator of sonic hedgehog pathway and a neural inducingsupplement to generate the MGE precursor cells. The pPS cells may becultured as an adherent culture or a suspension culture.

In certain embodiments, at the start of the method for production of MGEprecursor cells, pPS are plated cells into a cell culture container withan adherent substrate that facilitate the attachment of the pPS cellsand the cells are contacted with serum free medium comprising anactivator of sonic hedgehog pathway and a neural inducing supplement togenerate the MGE precursor cells.

In certain embodiments, the method may include culturing the pPS cellsin a serum free medium comprising an activator of sonic hedgehog pathwayand a neural inducing supplement to generate embryoid bodies (EBs),wherein the EBs comprise the MGE precursor cells.

In certain embodiments, at the start of the method for production of MGEprecursor cells, pPS may be plated cells in suspension in culturecontainers having a substrate with low adhesion properties that allowssuspension embryoid bodies to form. In an exemplary method, confluentmonolayer cultures of pPS cells are harvested and then plated innon-adherent cell culture plates, keeping the cells in suspension.

In certain cases, CollagenaseIV/Dispase may be used for preferentialselection for pPS colonies. The colonies may be trypsinized to singlecells and plated into low-attachment round-bottom plates to formsuspension EB.

In certain cases, the process of differentiation can be induced bycausing the pPS cells to differentiate, to form embryoid bodies oraggregates: for example, by overgrowth of a donor pPS cell culture, orby culturing pPS cells in suspension in culture vessels having asubstrate with low adhesion properties that allows embryoid bodies toform, or culturing pPS in absence of feeder layer. In an exemplarymethod, confluent monolayer cultures of pPS cells are harvested and thenplated in non-adherent cell culture plates, keeping the cells insuspension, and providing regular feeding with nutrient medium.

Alternatively or in addition, the differentiation process can beinitiated by culturing with certain factors that prevent the cells frommaintaining the undifferentiated phenotype. The initial differentiationfactors need not limit differentiation into the MGE precursor celllineage, but should be inclusive of MGE precursor cell or theirprecursors within the range of cell types in the differentiatedpopulation.

At some stage, the culture can be directed more specifically into theMGE precursor cell lineage. This can be done by including in the culturemedium a factor that more specifically promotes the generation andproliferation of MGE precursor cell, Exemplary factors that promote theformation and/or growth of MGE precursor cells include neural inducingsupplements as provided herein, activators of shh signaling, inhibitorsof BMP-signaling, inhibitors of TGF-β signaling, Wnt inhibitors, andanti-apoptotic agents, and in some cases can include activator(s) of FGFsignaling.

Exemplary methods for generating MGE precursor cells are describedbelow.

In certain cases, the method may include a step of generation of sEBfollowing by a step of generation of aEB. In other cases, the step ofgeneration of sEB may be replaced by an adherent culture.

Generation of Suspension Embryoid Bodies (sEB)

In an exemplary method, culturing pPS cells in suspension in culturevessels having a substrate with low adhesion properties that allowssuspension embryoid bodies to form may be carried out in the presence ofan activator of shh and a neural inducing supplement, such as B27 orNS21, The pPS cells may be cultured in suspension in absence of a feederlayer for 0 day-9 days before an activator of shh and/or neural inducingsupplement is added to the culture medium, for example, the pPS cellsmay be cultured in suspension for at least 0 hr, 1 hr, 3 hrs, 6 hrs, 12hrs, 18 hrs, 24 hrs, 36 hrs, 48 hrs, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, or 9 days before an activator of shh and/or neuralinducing supplement is added to the culture medium. Accordingly, the pPSare induced to form sEBs in the presence of a neural inducing supplementas described herein and an activator of shh signaling.

The pPS cell may be cultured in suspension to form sEB for a period ofat least 1 day, e.g., 1-100 days, 1-60 days, 1-50 days, 2-100 days, 2-50days, 3-100 days, 4-100 days, 5-10 days, or 7-10 days, or 25-100 days inthe presence of a neural inducing supplement as described herein and anactivator of shh signaling. In cases, where the pPS cell may be culturedin suspension to form sEB for a period of less than 9 days, an activatorof shh and/or neural inducing supplement may be added to the culturemedium within 0-8 days from the start of the culture of pPS cells toform sEB.

In certain embodiments, the pPS are plated in suspension, in culturecontainers having a substrate with low adhesion properties, in a cellculture medium that includes a neural inducing supplement as providedherein and an activator of shh signaling.

In addition to a neural inducing supplement as provided herein and anactivator of shh signaling, the culture medium for culturing pPS cellsin suspension to form sEBs may contain one or more of an anti-apoptoticagent, SMAD inhibitor (e.g., TGE-β inhibitors, BMP inhibitors, Activininhibitor, Nodal inhibitor, or growth differentiation factor (GDF)signaling pathway inhibitor), and Wnt inhibitor.

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes ananti-apoptotic agent, e.g., a ROCK inhibitor, for about 1 hr-35 days,e.g., at least 1 hr, at least 3 hrs, at least 10 hrs, at least 24 hrs,at least 36 hrs, at least 48 hrs, at least 2 days, at least 3 days, suchas, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 15 days, 20 days, 25days, or 35 days, An exemplary method may include plating the pPS cellsin suspension in a medium containing an anti-apoptotic agent, culturingthe pPS cells for a period of 1 hr-35 days in the presence of theanti-apoptotic agent. In certain cases, the anti-apoptotic agent may bepresent from the start of culturing of pPS cells in suspension and maybe removed after 1 hr-35 days, such as 1 day to 7 days, e.g. 1 days, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In certain cases, the anti-apoptotic agent may be present transientlyduring differentiation of the pPS into MAGE cells, e.g., theanti-apoptotic agent may be present in the culture medium on day 1 whenthe pPS cells are exposed to the neural inducing supplement as providedherein and an activator of shh signaling. The differentiation of the pPScell may be carried out in the presence of neural inducing supplement asprovided herein, an activator of shh signaling, and an anti-apoptoticagent for 1 hr to 35 days are noted above, after which the culturing maybe continued in the absence of the anti-apoptotic agent.

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes oneor more inhibitors of wnt. Although the Wnt signal inhibitor may beadded to the medium already at the start of cultivation of pPS cells, itmay be added to the medium after several days of cultivation (forexample, at a time within 10 days of cultivation). In certain cases, theWnt signal inhibitor is added to the medium at a time within 5 days ofstart of culturing of pPS cells in suspension, such as, within 0 days, 1day, or 3 days. The wnt inhibitor may be present throughout the step ofgeneration of sEB or may be present for a period of 5 days-10 days,e.g., 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, after whichthe culture may be continued in absence of the writ inhibitor(s).

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes oneor more inhibitors of SMAD. Although the SMAD signal inhibitor may beadded to the medium already at the start of cultivation of pPS cells, itmay be added to the medium after several days of cultivation (forexample, at a time within 10 days of cultivation). In certain cases, theone or more SMAD signal inhibitors are added to the medium at a timewithin 5 days of start of culturing of pPS cells in suspension, such as,within 0 days, 1 day, or 3 days. The wnt inhibitor may be presentthroughout the step of generation of sEB or may be present for a periodof 5 days-10 days.

In certain cases, the pPS are differentiated in the presence of shhactivator, neural inducing supplement as provided herein, and SMADinhibitor(s) for a period of 5 to 15 days (e.g., 5-10 days, such as, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days) after which thedifferentiation may be continued in absence of the SMAD inhibitor(s).

In certain embodiments, the method of producing medial ganglioniceminence (MGE) precursor cells from primate pluripotent stem (pPS) cellsmay include culturing the pPS cells in a serum free medium to generatesEBs, wherein the sEBs include the MGE precursor cells, wherein theserum free medium includes an activator of sonic hedgehog pathway, ananti-apoptotic agent, an inhibitor of SMAD, an inhibitor of Wnt and B27.The sEB produced by the methods described herein include a population ofMGE precursor cells.

In certain embodiments, the method of producing medial ganglioniceminence (MGE) precursor cells from primate pluripotent stem (pPS) cellsmay include culturing the pPS cells in a serum free medium as suspensionculture to generate the MGE precursor cells, wherein the serum freemedium includes an activator of sonic hedgehog pathway, ananti-apoptotic agent, an inhibitor of SMAD, an inhibitor of Wnt and B27.In certain cases, the sEB may be dissociated and plated as a monolayerto generate a monolayer that includes MGE precursor cells.

In certain cases, the sEBs may be dissociated and plated as a monolayerafter about 5 days from the beginning of the differentiation of pPScells. For example, the sEBs may dissociated and plated as a monolayerwithin 1-100 days after formation of the sEB, e.g., 1-75 days, 1-50days, 1-30 days, 1-10 days. In exemplary cases, sEB may be dissociatedand plated as a monolayer after about 10 days of formation of the sEB,e.g., 10-50 days, 10-40 days, 10-30 days, 10-20 days, 10 days, 12 days,etc. The differentiation factors as well as additives, supplements, orfactors, used as well as the timing of addition/removal of the same maybe as disclosed above.

In certain embodiments, the method of producing medial ganglioniceminence (MGE) precursor cells from primate pluripotent stem (pPS) cellsmay include culturing the pPS cells in a serum free medium as anadherent culture to generate the MGE precursor cells, wherein the serumfree medium includes an activator of sonic hedgehog pathway, ananti-apoptotic agent, an inhibitor of SMAD, an inhibitor of Wnt and B27.

In general, the MGE precursor cells produced by the method describedherein express a marker of MGE precursor cells, such as, NKX2.1.

In certain cases, the PS cells at the start of the culturing to generateMGE precursor cells are present at a cell density of 10³ to 10⁷cells/ml.

The medium used in the suspension culture can be prepared using anybasal medium. The medium may be BME medium, BGJb medium, CMRL 1066medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDMmedium, Medium 199 medium, Eagle's MEM medium, DMEM medium, Ham'smedium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, and amixed medium thereof and the like. The medium may be modified byaddition of additives, supplements, or factors, as disclosed herein.

A cell culture container with an adherent substrate may be used inmethods of culturing the pPS as an adherent culture. The differentiationfactors as well as additives, supplements, or factors, used as well asthe timing of addition/removal of the same may be as disclosed above.

Generation of Adherent Embryoid Bodies (aEB)

In certain cases, the sEBs generated by the above described methods maybe plated into a cell culture container with an adherent substrate thatfacilitate the attachment of the sEB to form adherent EBs. In general,the sEB may be plated onto a cell culture container with an adherentsubstrate in a culture medium containing a neural inducing supplement asprovided herein and an activator of shh signaling.

In embodiments where the pPS cells are cultured as an adhesion culture,as noted above, the method may further culturing the pPS cells in theserum free medium comprising the activator of sonic hedgehog pathway andthe neural inducing supplement to generate aEBs, which aEBs include MGEprecursor cells.

The sEB replated and cultured in adhesion culture to form aEB may becultured for a period of 1-100 days. In exemplary methods, the replatingof sEB may involve the steps of dissociating the EBs mechanically orenzymatically to produce single cells or clusters of cells, plating thedissociated cells on an adherent substrate to provide an adherentmonolayer; and culturing the adherent monolayer to generate aEBs. Incertain methods, the sEBs are not dissociated before further culturingin adherent conditions.

In certain embodiments, the method for generating MGE precursor cellsfrom pPS cells may include culturing the pPS cells in a serum freemedium comprising an activator of sonic hedgehog pathway and a neuralinducing supplement to generate sEBs and plating of the sEBs on a cellculture container with an adherent substrate and culturing the platedsEBs on the adherent substrate in the serum free medium comprising theactivator of sonic hedgehog pathway and the neural inducing supplementto generate aEBs, which aEBs include MGE precursor cells.

In certain cases, the aEBs may be dissociated and replated as amonolayer, which monolayer may be cultured in a serum free medium thatincludes the activator of sonic hedgehog pathway and the neural inducingsupplement to generate MGE precursor cells.

The cell culture medium for culturing of the adhesion culture togenerate aEB from the sEB may also include one or more of factors suchas, an anti-apoptotic agent, an inhibitor of SMAD, and an inhibitor ofWnt. The factors may be present at the start of the adhesion culture ormay be added within 5 days of initiation of the adhesion culture, suchas, 0 hr, 1 hr, 3 hr, 10 hr, 1 day, 2 days, or 3 days from theinitiation of the adhesion culture. The factors may be removed from theadhesion culture after 1 day to 20 days of culturing.

In certain embodiments, the method for generating MGE precursor cellsfrom pPS cells may include culturing the cells of the sEBs obtained bythe methods described herein in an adhesion culture in a medium thatincludes activator of sonic hedgehog pathway, a neural inducingsupplement, Wnt and SMAD inhibitors, for a period of 4-20 days, followedby culturing the adhesion culture for 8-20 days in a medium thatincludes activator of sonic hedgehog pathway and a neural inducingsupplement but does not include Wnt and SMAD inhibitors.

The aEBs generated by the methods described herein include a populationof MGE precursor cells. In general, the MGE precursor cells present inthe aEBs produced by the method described herein express NKX2.1 andFOXG1. In certain cases, the MGE precursor cells produced by the methodsdisclosed herein may express one or more markers of MGE precursor cells,such as, NKX2.1, LHX6, LHX7/8, FOXG1, OLIG2, DLX1/2, and ASCL1.

In certain embodiments, the MGE precursor cells produced by the methodsdescribed herein may also include a population of cells differentiatedfrom the MGE precursor cells, such as, interneurons, e.g., GABAergicinterneurons.

In general, the methods described herein result in generation of MGEprecursor cells at a high efficiency, resulting in cell cultures whereat least 50% (e.g. 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the cells inthe cell culture are MGE precursor cells.

As such, the method may include culturing pPS cells in a serum freeculture medium comprising activator of sonic hedgehog pathway and aneural inducing supplement for a period of 10-100 days (e.g. 5-50 days)to generate MGE precursor cells, wherein the pPS cells are cultured inadherent or suspension culture, wherein the OS cells are induced todifferentiate prior to culturing in the presence of activator of sonichedgehog pathway and a neural inducing supplement. As such, the pPScells may include differentiated cells, such as EBs, prior to culturingOS cells in a serum free culture medium comprising activator of sonichedgehog pathway and a neural inducing supplement. In certain cases, theserum free culture medium may additionally include an anti-apoptoticagent, an inhibitor of SMAD, and an inhibitor of Wnt.

In certain cases, the aEBs obtained from the sEB may be replated in asuspension culture to forma sEBs or dissociated and replated as amonolayer in adherent culture.

Culturing of pPS cells as an adherent culture in a method for generatingMGE precursor cells is further described below.

Adherent Culture for Generation of MGE Precursor Cells

As noted above, in certain embodiments, at the start of the method forproduction of MGE precursor cells, pPS are plated cells into a cellculture container with an adherent substrate that facilitate theattachment of the pPS cells and the cells are contacted with serum freemedium comprising an activator of sonic hedgehog pathway and a neuralinducing supplement to generate the MGE precursor cells.

In certain cases, the MGE precursor cells generated in the adherentculture may be present in the aEBs.

In some cases, the aEB produced by the subject culture method may bedissociated and replated as a monolayer and cultured in a serum freemedium comprising an activator of sonic hedgehog pathway and a neuralinducing supplement to generate the MGE precursor cells. The aEB may bemaintained in the of supplements and factors as described herein for aperiod of time of 1-100 days before being replated in a suspensionculture and cultured further as sEB or before being dissociated andreplated as a monolayer in an adherent culture. In certain cases, theperiod of time may be 1-75 days, 1-50 days, 1-30 days, 1-10 days, e.g.,10-50 days, 10-40 days, 10-30 days, 10-20 days, 5 days, 10 days, 20days, or 30 days.

Adherent substrates known in the art as well as those described hereinmay be used for culturing the pPS as an adherent culture in a method forgenerating MGE precursor cells.

The pPS cells may be grown as an adherent culture for a period of timebefore contacting with serum free medium comprising an activator ofsonic hedgehog pathway and a neural inducing supplement. In certaincases the pPS cells may be induced to differentiate by overgrowth of adonor pPS cell culture, or culturing pPS in absence of feeder layer, orculturing pPS cells in presence of FGF, or the like. Alternatively or inaddition, the differentiation process can be initiated by culturing withcertain factors that prevent the cells from maintaining theundifferentiated phenotype. The initial differentiation factors need notlimit differentiation into the MGE precursor cell lineage, but should beinclusive of MGE precursor cell or their precursors within the range ofcell types in the differentiated population.

At some stage, the culture can be directed more specifically into theMGE precursor cell lineage. This can be done by including in the culturemedium a factor that more specifically promotes the generation andproliferation of MGE precursor cell. Exemplary factors that promote theformation and/or growth of MGE precursor cells include neural inducingsupplements as provided herein, activators of shh signaling, inhibitorsof BMP-signaling, inhibitors of TGF-β signaling, Wnt inhibitors, andanti-apoptotic agents, and in some cases can include activator(s) of FGFsignaling.

Exemplary methods for generating MGE precursor cells are describedbelow.

The pPS cells may be cultured in adherent conditions for 0 day-9 daysbefore an activator of shh and/or neural inducing supplement is added tothe culture medium, for example, the pPS cells may be cultured inadherent conditions for at least 0 hr, 1 hr, 3 hrs, 6 hrs, 12 hrs, 18hrs, 24 hrs, 36 hrs, 48 hrs, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, or 9 days before an activator of shh and/or neuralinducing supplement is added to the culture medium. In certainembodiments, the pPS are differentiated in absence of a feeder celllayer.

In cases, where the pPS cell may be cultured in adherent conditions in aculture medium containing an activator of shh and/or neural inducingsupplement for a period of 1-100 days from the start of the culture ofpPS cells to form MGE precursor cells.

In addition to a neural inducing supplement as provided herein and anactivator of shh signaling, the culture medium for culturing pPS cellsin adherent conditions may contain one or more of an anti-apoptoticagent, SMAD inhibitor (e.g., TGF-β inhibitors, BMP inhibitors, Activininhibitor, Nodal inhibitor, or GDF signaling pathway inhibitor), and Wntinhibitor.

The timing of addition and removal of differentiation factors may be asdescribed for the aEB formation above.

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes ananti-apoptotic agent, e.g., a ROCK inhibitor, for about 1 hr-35 days,e.g., at least 1 hr, at least 3 hrs, at least 10 hrs, at least 24 hrs,at least 36 hrs, at least 48 hrs, at least 2 days, at least 3 days, suchas, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 15 days, 20 days, 25days, or 35 days. An exemplary method may include plating the pPS cellsin adherent culture in a medium containing an anti-apoptotic agent,culturing the pPS cells for a period of 1 hr-35 days in the presence ofthe anti-apoptotic agent. In certain cases, the anti-apoptotic agent maybe present from the start of culturing of pPS cells for generation ofMGE precursor cells and may be removed after 1 hr-35 days, such as 1 dayto 7 days.

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes oneor more inhibitors of wnt. Although the Writ signal inhibitor may beadded to the medium already at the start of culturing of pPS cells, itmay be added to the medium after several days of cultivation (forexample, at a time within 10 days of culturing). In certain cases, theWnt signal inhibitor is added to the medium at a time within 5 days ofstart of culturing of pPS cells in adherent condition, such as, within 0days, 1 day, or 3 days. The wnt inhibitor may be present throughout theculturing or may be present for a period of 5 days-10 days.

In certain cases, the method for producing MGE precursor cells from pPScells may include culturing the pPS cells in a medium that includes oneor more inhibitors of SMAD. Although the SMAD signal inhibitor may beadded to the medium already at the start of culturing of pPS cells, itmay be added to the medium after several days of culturing forgeneration of MGE precursor cells (for example, at a time within 10 daysof culturing). In certain cases, the one or more SMAD signal inhibitorsare added to the medium at a time within 5 days of start of culturing ofpPS cells in adherent condition, such as, within 0 days, 1 day, or 3days. The wnt inhibitor may be present throughout the culturing togenerate MGE precursor cells or may be present for a period of 5 days-10days.

In certain embodiments, the method of producing medial ganglioniceminence (MGE) precursor cells from primate pluripotent stem (pPS) cellsmay include culturing the pPS cells in adherent condition to generateMGE precursor cells, wherein the culture medium includes an activator ofsonic hedgehog pathway, an anti-apoptotic agent, an inhibitor of SMAD,an inhibitor of Wnt and B27.

In certain cases, the PS cells at the start of the culturing to generateMGE precursor cells are present at a cell density of 10³ to 10⁷cells/ml.

As noted above, a serum free medium may be used in the method ofgenerating MGE precursor cells from pPS cells, A serum-free medium meansa medium not containing an unadjusted or unpurified serum, such as,fetal bovine serum, fetal calf serum. The serum-free medium may includea serum replacement, such as, those described herein, e.g., B27 or NS21.

Culture of MGE Precursor Cells

The sEBs and aEBs generated by the methods described herein may bedissociated, enzymatically or mechanically, and cultured as a monolayeron a cell culture vessel with adherent substrate. Accordingly, the MGEprecursor cells present in the sEB and aEBs may be cultured as amonolayer.

In certain cases, culturing of MGE precursor cells in a monolayer may becarried out for a period of 1-100 days, such as 10 days-15 days.

The culturing of MGE precursor cells in a monolayer may be carried outin a culture medium that contains a neural inducing supplement asprovided herein and an activator of shh signaling.

In certain cases, the MGE precursor cells generated by the methoddescribed herein may be cultured in a culture medium that promotegeneration of neurons, such as, inhibitory interneurons, e.g., GABAergicinterneurons. Accordingly, sEB, aEB, and monolayer produced fromdissociation of sEBs and aEBs generated by the methods described hereinand containing MGE precursor cells may be contacted with a culturemedium that promotes differentiation of the MGE precursor cells intopost mitotic neurons. In certain cases, this culture medium may notinclude SMAD inhibitors. In addition, in certain cases, this culturemedium may include SMAD activators. As such, the culture medium mayinclude SMAD activators in order to increase the population ofinterneurons present in the MGE precursor cells generated by theprotocols described herein. Exemplary SMAD activators include TGFs(e.g., TGFβ3), BMPs (e.g., BMP2, BMP4, BMP8), Activin, Nodal, GDF, andIDE1.

In certain cases, the culture medium to promote differentiation of theMGE precursor cells to interneurons may include a NOTCH inhibitor, BDNF,GDNF, NT3, NT4, camp, vitamin c, serum, matrigel, insulin, IGF, SDF1a,Neuregulin1, TGFβ.

The culturing of MGE precursor cells in a monolayer may lead toproliferation of MGE precursor cells and/or differentiation of MGEprecursor cells into cells having a neuronal cell fate. In certaincases, the MGE precursor cells that differentiate into cells having aneuronal cell fate express DLX1/12, TUJ, MAP2, GAD1/2, and GABA, and mayexpress one or more of NKX2.1, ASCL1, LHX6, LHX7/8, DCX, NEUN, and VGAT,and may express subtype markers calbindin, calretinin, somatostatin, andpare albumin.

In certain cases, the MGE precursor cells generated by the methoddescribed herein may be co-cultured with a support cell population toinduce differentiation of the MGE precursor cells into interneurons,such as, GABAergic interneurons.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation without promoting differentiation.Exemplary ES medium is made with 80% DMEM (such as Knockout DMEM “KODMEM”), 20% of either defined fetal bovine serum (FBS, Hyclone) or serumreplacement (e.g., knockout serum replacement (KSR)), 1% non-essentialamino acids (NEAA), 1% pen-step-glutamine (1 mM L-glutamine), 0.0008%β-mercaptoethanol, and 10 ng/ml FGF-basic (bFGF).

The pPS cells can be expanded in the undifferentiated state by culturingin an environment that inhibits differentiation. Traditionally, pPScells are cultured on a layer of feeder cells derived from embryonic orfetal tissue of the mouse. Culture plates are plated with 375,000irradiated mouse embryonic fibroblasts (mEFs) per well (irradiated toinhibit proliferation but permit synthesis of factors that support pPScells), and used 5 h to 10 days after plating. In certain embodiments,human feeder cells may also be used.

pPS cells can be maintained in an undifferentiated state even withoutfeeder cells. The environment for feeder-free cultures includes asuitable culture substrate, particularly an extracellular matrix such asMatrigel® or laminin. The pPS cells are plated at >15,000 cells cm⁻²(optimally 90,000 cm⁻² to 170,000 cm⁻²). Feeder-free cultures aresupported by a nutrient medium containing factors that supportproliferation of the cells without differentiation. Such factors may beintroduced into the medium by culturing the medium with cells secretingsuch factors, such as irradiated (˜4,000 rad) primary mouse embryonicfibroblasts, telomerized mouse fibroblasts, or human feeder cellsderived from pPS cells. Medium can be conditioned by plating the feedersat a density of 5-6×10⁴ cm⁻² in a serum free medium such as KO DMEMsupplemented with 20% serum replacement and 4 to 8 ng/mL bFGF. Mediumthat has been conditioned for 1-2 days is supplemented with furtherbFGF, and used to support pPS cell culture for 1-2 days. Features of thefeeder-free culture method are further discussed in International PatentPublications WO99/20741 & WO01/51616; and Xu et al., Nat. Biotechnol,19:971, 2001, which are herein incorporated by reference.

Factors

The methods and compositions of the present disclosure involve the useof various factors, such as, neural inducing supplements, anti-apoptoticagents, differentiation factors, and the like. Examples of neuralinducing supplements, anti-apoptotic agents, differentiation factorsused in the methods and compositions of the present disclosure aredescribed below.

Neural Inducing Supplement

Exemplary neural inducing supplements include B27, NS21, or anequivalent supplement.

In certain embodiments, the neural inducing supplement may be B27, B-27®Serum-Free Supplement is available from Life Technologies. B27supplement contains bovine serum albumin, transferrin, insulin,progesterone, corticosterone, triiodo-1-thyronine, retinol acetate, DLtocopherol, DL tocopherol acetate, Biotin, Linoleic acid, Linolenicacid, ethanolamine, Na Selenite, L-carnitine, glutathione reduced,catalase, superoxide dismutase, D-galactose and putrescine. In certaincases, B27-vitamin A may be used.

In certain cases, the neural inducing supplement may be NS21. NS21 isdescribed in Y, Chen et al., J. Neurosci. Methods., 171:239, 2008. Y.Chen et al. showed that NS21 is equivalent to B27 supplement in aneuronal culture. The formulation of NS21 is described in Y, Chen et al.and is reproduced in Table 1 below,

TABLE 1 NS1 Formulation For 400 ml Stock NS21 (20 L μg/ml μM (mg/ml)final medium) Final Concentration Albumin, bovine 2500 37 Add as 50 gpowder Catalase 2.5 0.010 Add as 50 mg powder Glutathione 1.0 3.2 Add as20 mg (reduced) powder Insulin 4.0 0.6 10 8 ml Superoxidase 2.5 0.077Add as 50 mg dismutase powder Holo transferrin 5.0 0.062 Add as 100 mgpowder T3 (triiodol-1- 0.002 0.0026 2.0 20 μl thyronin) L-Carnitine 2.012 Add as 40 mg powder Ethanolamine 1.0 16 Liquid 20 μl (1 g/ml)D(+)-galactose 15 83 Add as 300 mg powder Putrescine 16.1 183 Add as 322mg powder Sodium Selenite 0.01435 0.083 1.0 280 μl Ethanolic StocksCorticosterone 0.02 0.058 2.0 0.2 ml Linoleic acid 1.0 3.5 100.0 0.2 mlLinolenic acid 1.0 3.5 100.0 0.2 ml Lipoic acid 0.047 0.2 4.7 0.2 ml(thioctic acid) Progesterone 0.0063 0.020 3.2 0.04 ml Retinol acetate0.1 0.2 20.0 0.1 ml Retinol, all trans 0.1 0.3 10.0 0.2 ml (vit. A) D,L-alpha-Tocoph- 1.0 2.3 100.0 0.2 ml erol (vit. E) D, L-alpha-Tocoph-1.0 2.1 100.0 0.2 ml erol acetate

In certain cases, the neural inducing supplement may be present in theserum free medium for culturing pPS cells at a concentration rangingfrom 0.5% to 10%, for example, 0.5%-5%, e.g., 0.5%, 1%, 2%, or 3%.

In certain embodiments, the serum free medium comprising a shh activatorand a neural inducing supplement for culturing of pPS to generate EBsdoes not include KSR or N2 supplement. In certain embodiments, themethod of generating MGE precursor cells does not include culturing thepPS cells in a serum free medium comprising bFGF or FGF-2. In certaincases, the pPS cells are cultured in a serum free medium comprising ashh activator and a neural inducing supplement and not containing KSR orN2 supplement or bFGF or FGF-2 for a period of time sufficient togenerate sEB or aEB.

In certain cases, the sEBs may be further cultured in a serum freemedium comprising a shh activator and a neural inducing supplement andfurther containing one or more of KSR supplement, N2 supplement, bFGF,and FGF-2 for a period of sufficient to generate aEB.

In certain cases, the pPS cells may be cultured in a serum free mediumcomprising a shh activator and a neural inducing supplement and furthercontaining one or more of KSR supplement, N2 supplement, bFGF, and FGF-2for a period of sufficient to generate sEB. The additional supplementsmay be added at the same time as a shh activator and the neural inducingsupplement as described herein or at a later time point, such as, after5 days 2 weeks, such as, after 1 weeks-2 weeks after exposing the pPScells to shh activator and the neural inducing supplement as describedherein. In certain cases, the KSR supplement and/or N2 supplement may bepresent added at day 0 of differentiation, or later such as day 5, day7, day 10, day 14, day 21, after contacting the pPS cells shh activatorand the neural inducing supplement as described herein.

In certain embodiments, the cell culture medium used in the methodsdisclosed herein does not include serum replacements, such as, KSR orN2.

Anti-Apoptotic Agents

In certain embodiments of the methods and compositions described herein,an anti-apoptotic agent may be included in the medium for PS culturingcells.

In certain cases, the anti-apoptotic agent may be an inhibitor ofRho-associated protein kinase (ROCK). In certain cases, the ROCKinhibitor may be Y27632, HA-100, H-1152,(+)-trans-4-(1-aminoethyl)-1-(pyridin-4-ylaminocarbonyl) cyclohexanedihydro-chloride monohydrate (described in WO00078351, WO00057913),imidazopyridine derivatives (described in U.S. Pat. No. 7,348,339),substituted pyrimidine and pyridine derivatives (described in U.S. Pat.No. 6,943,172) and substituted isoquinoline-sulfonyl compounds(described in EP00187371), or GSK429286A, ROCKII inhibitor, orThiazovivin, or an analog or derivative thereof.

The anti-apoptotic agent may be present at a concentration of 0.1 μM,0.3 μM, 0.5 μM, 1 μM, at least about 1.3 μM, at least about 1.5 μM, atleast about 2 μM, at least about 2.3 μM, at least about 2.5 μM, at leastabout 2.8 μM, at least about 3 μM, at least about 3.5 μM, at least about4 μM, at least about 4.5 μM, at least about 5 μM, at least about 10 μM,at least about 20 μM, at least about 30 μM, at least about 40 μM or atleast about 50 μM, such as, 0.5 μM-50 μM, 1 μM-25 μM, or 2.5-20 μM.

Inhibitors of SMAD

In certain embodiments of the methods and compositions described herein,an inhibitor of SMAD may be present in the medium for culturing cells.In some embodiments, an inhibitor of SMAD can be present in the medium,used for culturing cells, at a concentration of 10 ng/ml, 200 ng/ml, 300μg/ml, 400 ng/ml, 500 ng/ml, 1 μg/ml, 1.5 μg/ml, 1, 2 μg/ml, 2.5 μg/ml,or 5 μg/ml for example, at a concentration of 500 ng/ml-3 μg/ml, 1μg/ml-3 μg/ml.

The inhibitor of SMAD may be present at a concentration of at leastabout 0.01 μM, at least about 0.03 μM, at least about 0.1 μM, at leastabout 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at leastabout 1 μM, at least about 1.3 μM, at least about 1.5 μM, at least about2 μM, at least about 2.3 μM, at least about 2.5 μM, at least about 2.8μM, at least about 3 μM, at least about 3.5 μM, at least about 4 μM, atleast about 4.5 μM, at least about 5 μM, at least about 10 μM, at leastabout 20 μM, at least about 30 μM, at least about 40 μM or at leastabout 50 μM, such as, 0.5 μM-50 μM, 1 μM-25 μM, or 5 μM-20 μM.

In certain embodiments, the inhibitor of SMAD may be an inhibitor ofTGF-β signaling. For example, the SMAD inhibitor may be an ALKinhibitor, or antibody or a fragment thereof that binds to TGF-β1,TGF-β2, TGF-β3, TGF-β receptor I and/or II. In certain embodiments, theinhibitor of TGF-β signaling may be a small molecule inhibitor. Incertain cases, the inhibitor of TGF-β signaling may be LY364947 (SD208),SM16, SB-505124, ALK5 Inhibitor II, SB-431542, LY2157299, LDN-193189,A83-01, (+)-ITD-1 ITD-1 (ethyl4-([1,1′-biphenyl]-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate),or ITDts.

In certain embodiments, the SMAD inhibitor may be BMPRIA-Fc, Noggin, orderivatives thereof.

In certain embodiments, the SMAD inhibitor may be a BMP pathwayinhibitor, such as, dorsomorphin.

In certain embodiments, the SMAD inhibitor may be an Activin inhibitor,Nodal inhibitor, or GDF signaling pathway inhibitor. Exemplary activininhibitors include SB431542, Follistatin, A8301, DMH1, Dorsomorphin,K02288, and SB505124. In certain cases, inhibitors of Nodal, such as,SB431542, Lefty, or Cerebrus may be used. In certain cases, SB431542,D4476, GW788388, LY364947, RepSox, SB525334, SD208 may be used toinhibit GDF signaling pathway.

In certain embodiments, two or more SMAD inhibitors may be included inthe cell culture medium used in the methods described herein.

Activators of Sonic Hedgehog Signaling

In certain embodiments of the methods and compositions described herein,an activator of sonic hedgehog signaling may be present in the mediumfor culturing cells. The activator of sonic hedgehog signaling may bepresent at a concentration of at least about 0.01 μM, at least about0.03 μM, at least about 0.1 μM, at least about 0.2 μM, at least about0.25 μM, at least about 0.3 μM, at least about 1 μM, at least about 1.3μM, at least about 1.5 μM, at least about 2 μM, at least about 2.3 μM,at least about 2.5 μM, at least about 2.8 μM, at least about 3 μM, atleast about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at leastabout 5 μM, at least about 10 μM, at least about 20 μM, at least about30 μM, at least about 40 μM or at least about 50 μM, such as, 0.05 μM-5μM, 0.01 μM-2.5 μM, 0.05 μM-2 μM, or 0.1 μM-2 μM.

In certain cases, the activator of sonic hedgehog signaling may be shhor a derivative thereof. In certain cases, the activator of sonichedgehog signaling may be a small molecule, such as, purmorphamine, SAGsmoothened agonist, Hh-Ag1.5, or derivatives and analogs thereof.

Wnt Inhibitor

In certain embodiments of the methods and compositions described herein,an inhibitor of Writ signaling may be present in the medium forculturing cells.

Wnt inhibitors are agents that downregulate expression or activity ofwnt. Agents of interest may interact directly with wnt, e.g. drugs,i.e., small molecules, blocking antibodies, etc., or may interact withwnt associated proteins, e.g. Wnt co-receptors LRP5/6 and thetransmembrane protein Yemen. A number of wnt inhibitors have beendescribed and are known in the art.

Wnt inhibitors of interest interfere with the interaction betweensoluble, extracellular Wnt proteins, and the frizzled receptors that arepresent on the surface of normal cells. Such agents include, withoutlimitation, soluble frizzled polypeptides comprising the wnt bindingdomains; soluble frizzled related polypeptides; wnt specific antibodies;frizzled specific antibodies; and other molecules capable of blockingextracellular wnt signaling.

Among the known wnt inhibitors are members of the Dickkopf (Dkk) genefamily (see Krupnik et al. (1999) Gene 238(2):301-13). Members of thehuman Dkk gene family include Dkk-1, Dkk-2, Dkk-3, and Dkk-4, and theDkk-3 related protein Soggy (Sgy).

Other inhibitors of wnt include Wise (Itasaki et al. (2003) Development130(18):4295-30), which is a secreted protein. The Wise proteinphysically interacts with the Wnt co-receptor, lipoproteinreceptor-related protein 6 (LRP6), and is able to compete with Wnt8 forbinding to LRP6.

Inhibitors may also include derivatives, variants, and biologicallyactive fragments of native inhibitors.

In certain cases, the Wnt inhibitor may be a small molecule such as,CKI-7, IWP analogs, IWR analogs, XAV939, 53AH, Wnt-C59.

In certain cases, the Wnt inhibitor may be present in the culture mediumat a concentration of 10 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500ng/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml, 2.5 μg/ml, or 5 μg/ml for example,at a concentration of 500 ng/ml-3 μg/ml, e.g., 1 μg/ml-3 μg/ml.

Assessing Generation of Cell Populations

In certain cases, the cell populations cultured according to the methodsdisclosed herein may be monitored to assess changes in the cellsimparted by culturing (e.g., during one or more time points in theculture method disclosed herein) so as to characterize the cellpopulation produced. In certain embodiments, the production of MGEprecursor cells (mitotic MGE precursor cells and/or post-mitoticinterneurons) may be assessed by determining the expression of markerscharacteristic of these cell populations.

In certain cases, the expression of certain markers is determined bydetecting the presence or absence of the marker transcript or proteinexpression. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such processes, themeasurement of marker expression can be qualitative or quantitative. Onemethod of quantitating the expression of markers that are produced bymarker genes is through the use of quantitative PCR (Q-PCR). Methods ofperforming Q-PCR are well known in the art. Other methods which areknown in the art can also be used to quantitate marker gene expression.For example, the expression of a marker gene product can be detected byusing antibodies specific for the marker gene product of interest. Incertain processes, the expression of marker genes characteristic of thecell population of interest as well as the lack of significantexpression of marker genes characteristic of PS cells and other celltypes may be determined.

Monitoring of generation of MGE precursor cells may be by determiningexpression of NKX2.1 gene. As such, the MGE precursor cells produced bythe processes described herein express the NKX2.1 marker gene, therebyproducing the NKX2.1 gene product. The MGE precursor cells produced bythe methods described herein also express the FOXG1, and may expressLHX7/8, OLIG2, ASCL1, and DLX2. Furthermore, the MGE precursor cellsproduced by the methods described herein do not express PAX6.

In some embodiments described herein, the expression of the NKX2.1marker and/or the FOXG1 marker in MGE precursor cells is at least about2-fold higher to at least about 10,000-fold higher than the expressionof the NKX2.1 marker and/or the FOXG1 marker in non-MGE precursor cells,for example pluripotent stem cells. In other embodiments, the expressionof the NKX2.1 marker and/or the FOXG1 marker in MGE precursor cells isat least about 4-fold higher, at least about 6-fold higher, at leastabout 8-fold higher, at least about 10-fold higher, at least about15-fold higher, at least about 20-fold higher, at least about 40-foldhigher, at least about 80-fold higher, at least about 100-fold higher,at least about 150-fold higher, at least about 200-fold higher, at leastabout 500-fold higher, at least about 750-fold higher, at least about1000-fold higher, at least about 2500-fold higher, at least about5000-fold higher, at least about 7500-fold higher or at least about10,000-fold higher than the expression of the NKX2.1 marker and/or theFOXG1 marker in non-MGE precursor cells, for example pluripotent stemcells.

In certain cases, the monitoring of generation of MGE precursor cells(mitotic MGE precursor cells and/or post-mitotic interneurons) may becarried out by performing functional analysis of the cells of interest.For example, MGE precursor cells generated by the methods describedherein may be may generate interneurons in vivo or in vitro. In certaincases, MGE precursor cells produced by the methods disclosed herein maygenerate interneurons that differentiate into inhibitory GABAergicinterneurons that can migrate and functionally integrate with neuron invivo.

In certain cases, the method does not include monitoring of generationof MGE precursor cells.

Enrichment, Isolation and/or Purification of Cell Populations

Cell populations of interest, such as, MGE precursor cells (mitotic MGEprecursor cells and/or post-mitotic interneurons) produced by any of theabove-described processes can be enriched, isolated and/or purified byusing an affinity tag that is specific for such cells. Examples ofaffinity tags specific for a cell or cell population of interest includeantibodies, ligands or other binding agents that are specific to amarker molecule, such as a polypeptide, that is present on the cellsurface of the cells of interest but which is not substantially presenton other cell types that may be found in a cell culture produced by themethods described herein.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and cells described herein. In one process, an antibody whichbinds to a marker expressed by cell population of interest is attachedto a magnetic bead and then allowed to bind to the cells of interest ina cell culture which has been enzymatically treated to reduceintercellular and substrate adhesion. The cell/antibody/bead complexesare then exposed to a magnetic field which is used to separatebead-bound definitive endoderm cells from unbound cells. Once the cellsof interest are physically separated from other cells in culture, theantibody binding is disrupted and the cells are replated in appropriatetissue culture medium.

Additional methods for obtaining enriched, isolated, or purified cellpopulations of interest can also be used. For example, in someembodiments, an antibody for a marker expressed by the cells of interestis incubated cell culture containing the cells of interest that has beentreated to reduce intercellular and substrate adhesion. The cells arethen washed, centrifuged and resuspended. The cell suspension is thenincubated with a secondary antibody, such as an FITC-conjugated antibodythat is capable of binding to the primary antibody. The cells are thenwashed, centrifuged and resuspended in buffer. The cell suspension isthen analyzed and sorted using a fluorescence activated cell sorter(FACS). Antibody-bound cells are collected separately from cells notbound to the marker specific antibody, thereby resulting in theisolation of cells of interest. If desired, the isolated cellcompositions can be further purified by using an alternateaffinity-based method or by additional rounds of sorting using the sameor different markers that are specific for the cells of interest. Incertain cases, the MGE precursor cells may be enriched by sorting thecells based on size.

In certain cases, cells of interest, such as, MGE precursor cells areenriched, isolated and/or purified from other types of cells after thePS cell cultures are induced to differentiate towards the MGE precursorcell lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the above-described procedures, cells of interest, suchas, MGE precursor cells may also be isolated by other techniques forcell isolation. Additionally, cells of interest, such as MGE precursorcells, may also be enriched or isolated by methods of serial subculturein growth conditions which promote the selective survival or selectiveexpansion of the cells of interest.

Using the methods described herein, cell populations or cell culturesenriched in cells of interest, such as, MGE precursor cells, by at leastabout 2- to about 1000-fold as compared to un-enriched cell populationsare produced. In some embodiments, MGE precursor cells can be enrichedby at least about 5- to about 500-fold as compared to untreated cellpopulations or cell cultures. In other embodiments, MICE precursor cellscan be enriched from at least about 10- to about 200-fold, at leastabout 20- to about 100-fold, at least about 40- to about 80-fold, or atleast about 2- to about 20-fold as compared to undifferentiated cellpopulations or cell cultures.

Genotypic Features of Cell Populations of the Present Disclosure

When derived from an isolated PS cell, or an established line of PScells, the cell populations of this disclosure can be characterized asbeing the progeny of the originating cell or cell line. Accordingly, thecell populations will have the same genome as the cells from which theyare derived. This means that over and above any karyotype changes, thechromosomal DNA will be over 90% identical between the PS cells and thecell populations generated therefrom. Cell populations of the presentdisclosure that have been treated by recombinant methods to introduce atransgene or knock out an endogenous gene are still considered to havethe same genome as the line from which they are derived, since allnon-manipulated genetic elements are preserved. Cell populations of thepresent disclosure and PS cells can be identified as having the samegenome by standard genetic techniques. Possession of the same genome canalso be inferred if the cell populations are obtained from theundifferentiated line through the course of normal mitotic division.

In certain industrial applications, this characteristic is a valuablefeature of the cell populations of the present disclosure. Inparticular, the availability of the originating PS cells provides afurther supply of genetically matched differentiated cell populations,since the PS cells can be caused to proliferate and differentiated intomore cell populations of the present disclosure as required.Furthermore, the PS cells can be differentiated into othertherapeutically important lineages.

The techniques described in this application allow for the production oflarge cell populations that share the same genome, by expanding thecells before or after differentiation. Populations of 10⁸, 10¹⁰, or 10¹²cells are theoretically possible. Such large populations are usuallydivided into separate containers suitable for further culture, drugscreening, or therapeutic administration.

Certain embodiments of the disclosure include originating cells (such asan undifferentiated PS cell line, or an intermediate population, incombination with one or more populations of differentiated cells bearingcharacteristics of MGE precursor cells. The populations may either be inthe same container, in separate containers in the same facility, or intwo different locations. The undifferentiated and differentiated cellsmay be present simultaneously or at a different time, such as when aculture of undifferentiated cells is caused to differentiate into MGEprecursor cells, as described herein.

Compositions Comprising Cell Populations of the Present Disclosure

Cell compositions produced by the above-described methods include cellcultures that contain isolated MGE precursor cells and cell populationsenriched in isolated MGE precursor cells.

In some embodiments, cell compositions which include cells of thepresent disclosure, wherein at least about 50%-80% of the cells inculture are the cells of interest, can be produced. The differentiationmethods described herein can result in about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or greater than about 95% conversion ofpluripotent cells to cells of interest.

In embodiments, in which isolation of cells of interest is employed, forexample, by using an affinity reagent that binds to the cells ofinterest, a substantially pure cell population of interest can berecovered.

Some embodiments described herein relate to cell compositions comprisingfrom at least about 5% cells of interest to at least about 95% cells ofinterest. In some embodiments, the cell cultures or cell populationscomprise mammalian cells. In preferred embodiments, the cell cultures orcell populations comprise human cells. For example, certain specificembodiments relate to cell compositions comprising human cells, whereinfrom at least about 5% to at least about 95% of the human cells are MGEprecursor cells. Other embodiments relate to cell compositionscomprising human cells, wherein at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90% or greater than 90% of the humancells are MGE precursor cells.

In a specific embodiment, a composition comprising human cells, where atleast 70% of the human cells are human MGE precursor cells is provided.

In another embodiment, a composition comprising human cells, where atleast 80% of the human cells are human MGE precursor cells is provided.

In certain embodiments, the composition may include pluripotent stemcells and/or inhibitory interneurons.

Cell populations of the present disclosure, such as, sEB comprising MGEprecursor cells, aEB comprising MGE precursor cells, MGE precursorcells, or neurons generated from MGE precursor cells may be present in acomposition comprising one or more of these cell populations.

The cells populations of the present disclosure may be used fresh orstored in art accepted methods, such as, cryopreserved for a period of 1day to 10 years before being thawed and used.

Cell compositions produced by the above-described methods andcompositions thereof may be assessed by using the markers and methodsdescribed herein as well as those known in the art.

Cell compositions produced by the above-described methods andcompositions thereof may be enriched, isolated or purified using methodsdescribed herein as well as those known in the art.

Uses of Populations of the Resent Disclosure

Cell Populations for Screening

The cells of the present disclosure can be used to screen for agents(such as, small molecules, peptides, polynucleotides) or environmentalconditions (such as, culture conditions or manipulation) that affect thecharacteristics of MGE precursor cells and/or cells generated therefrom,such as, interneurons.

In one example, MGE precursor cells are used to screen factors thatpromote maturation into interneurons, or promote proliferation andmaintenance of MGE precursor cells in tong-term culture. For example,candidate differentiation factors or growth factors are tested by addingthem to cells in different wells, and then determining any phenotypicchange that results, according to desirable criteria for further cultureand use of the cells. This can lead to improved derivation and culturemethods for generating and/or maintaining MGE precursor cells and/orcells generated therefrom.

Other screening methods of the present disclosure relate to the testingof pharmaceutical compounds for a potential adverse effect on MGEprecursor cells and/or cells generated therefrom. This type of screeningis appropriate not only when the compound is designed to have apharmacological effect on MGE precursor cells themselves, but also totest for MGE precursor cells-related side-effects of compounds designedfor a primary pharmacological effect elsewhere.

Other screening methods relate to the use of MGE precursor cells tomeasure the effect of small molecule drugs that have the potential toaffect MGE precursor cells. To this end, the cells can be combined withtest compounds in vitro, and the effect of the compound on MGE precursorcells is determined.

General principles of drug screening are described in U.S. Pat. No.5,030,015, and in the textbook In vitro Methods in PharmaceuticalResearch, Academic Press 1997. Assessment of the activity of candidatepharmaceutical compounds generally involves combining the differentiatedcells of this invention with the candidate compound, either alone or incombination with other drugs. The investigator determines any change inthe morphology, marker, or functional activity of the cells that isattributable to the compound (compared with untreated cells or cellstreated with a negative control compound), and then correlates theeffect of the compound with the observed change.

MGE Precursor Cells in Clinical Therapy

Cell populations comprising MGE precursor cells, such as, cellpopulations enriched in MGE precursor cells, as well as, purified MGEprecursor cells produced by the methods described herein may be used ina number of clinical applications.

In certain embodiments, the MGE precursor cells produced using themethods provided herein may be used for treating a subject in need fortreatment with MGE precursor cells.

In certain cases, a subject in need for treatment with MGE precursorcells may be a patient having or at risk of developing a neurologicaldisorder characterized by decreased inhibitory interneuron activity. Incertain cases, the patient may have reduced inhibitory neuron functionand/or elevated excitatory neuron function.

In certain cases, the MGE precursor cells may be transplanted into atarget site in the subject that provides appropriate differentiationconditions for the MGE precursor cells to differentiate intointerneurons, such as, GABAergic inhibitory interneurons, Cells may betransplanted by any of a number of standard methods in the art fordelivering cells to tissue, e.g., injecting them as a suspension in acarrier, such as, a suitable solution or a solid or semi-solid support.Suitable solutions include saline, PBS, L15, DMEM, Iscove's media, etc.Suitable solid supports include beads, a filter such as a mesh filter, amembrane, etc.

In certain cases, the MGE precursor cells may be administered to thenervous system of the subject. In certain cases, the administering maybe performed by transplanting the MGE precursor cells into one or morelocations in the nervous system of the subject.

In certain embodiments, the MGE precursor cells may be administered intoone or more locations in the nervous system of the subject, such as,central nervous system, such as, brain, e.g., cerebellum, cerebralcortex, hippocampus, striatum (e.g., basal ganglia), thalamus,hypothalamus, subthalamic nucleus; and spinal cord.

In certain embodiments, the administering of MGE precursor cells mayresult in the inhibitory neuron function being restored. In certaincases, the administering may include transplanting the MGE precursorcells in a first portion of the brain of the subject and restoringinhibitory neuron function in a second portion of the brain, distal fromthe first.

In certain embodiments, the MGE precursor cells may be administered to asubject having or at risk of developing a neurological disorder, suchas, seizure disorder, e.g., epilepsy, Huntington's disease, Parkinson'sdisease, ALS, schizophrenia, Alzheimer's disease, autism, dyskinesia,chronic pain, spasticity, neuropathic pain, multiple sclerosis,traumatic brain injury, diseases of dis-myelination, bi-polar disorder,depression, and cancer.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

-   -   Materials and Methods:    -   Cell Culture and FACS-Sorting

HES-3 hESCs were maintained on irradiated mouse embryonic fibroblasts(Millipore) in knockout DMEM with 20% knockout serum replacement, 1%nonessential amino acids (NEAA), 1% pen-strep-glutamine, 0.0008%2-mercaptoethanol, and 10 ng/mL FGF-basic (Invitrogen), Differentiationwas initiated by CollagenaseIV/Dispase (1 mg/mL each; Invitrogen)preferential selection for hESC colonies. Colonies were trypsinized tosingle cells, and, as described (Eiraku, M., et al. (2008). Cell StemCell 3, 519-532), ˜10,000 cells/well were plated into low attachmentround-bottom 96-well plates (Corning or NOF) to form one sEB/well inoptimized B27+5F differentiation medium #1, consisting of Neurobasal-A,2% B27-vitamin A (Invitrogen), and the same supplements in hESC mediabut without FGF, Also, Y27632 (100), SB431542 (10 μM), Purmorphamine(1-2 μM) (Stemgent), BMPRIA-Fc (1.5 μg/mL), and DIKK1 (1 μg/mL)(Invitrogen) were added.

On ˜d7, Y27632 was removed, and sEBs were plated adherent en-bloc ontomatrigel (BD Biosciences) coated plates in medium #1. On ˜d14, factorswere removed from medium #1 except for Purmorphamine. On ˜d25, aEBs weretrypsinized and replated as dissociated monolayer onto matrigel orpolyornithine/laminin coated plates. DAFT (10 μM) (Tocris) was addedfrom ˜d27-d30. After FACS sorting on ˜day 35, GFP+ cells could bereplated (10-25,000 cells/cm2) onto cortical glial cells in medium #1,Glial cells were prepared from newborn mouse cortex, passaged at least3× with serum to remove mouse neurons, and pre-treated at confluencywith Ara-C (5 μM) (Sigma). Every 3-4 days, half of media was replacedwith differentiation medium #2: Neurobasal-A, 2% B27+vitaminA,(Invitrogen) and same supplements in medium #1, without factors, exceptNEAA and Purmorphamine were removed, and BDNF (25 ng/mL) was added (R&DSystems). Media was replaced every 3-4 days.

FACS analysis and sorting was performed on FACS Aria II (BDBiosciences). Cells were gated for live cell DAN exclusion, smallscatter size, single cells, NKX2.1-GFP signal, and PSA-NCAM-APC or -PE(Miltenyi Biotech) signal intensity. GFP-negative cells and isotypeantibody (Santa Cruz) controls were used. Data was analyzed with Flowjo(Treestar) software. For live cell imaging, differentiation was similarto above, but 5,000 cells/well were plated into Aggrewell-800 plates(Stem Cell Technologies), and sEBs were plated en-bloc on day 4-7.Imaging was performed by time-lapse confocal microscopy with temperature(37° C.) and gas (5% O2, 5% CO2, 90% N2) controls (Leica SP5).

Second trimester human fetal cortex or MGE tissue was dissected,dissociated to single cells with Papain (Worthington Biochemical),plated onto matrigel coated plates (˜100,000 cells/cm²) indifferentiation medium #2, and treated with Ara-C at confluence. MGEcells cultured for one week, or hESCs sorted on day 35, were replatedonto cortical cultures (˜10-20,000 cells/cm²).

All experiments conducted on hESCs and human fetal tissue adhered toapproved UCSF Stem Cell Research Oversight committee and Committee onHuman Research protocols.

Transplantation and Graft Proliferation

Excess medium was removed from sorted cell pellets to createconcentrated cell suspensions of 1000 cells/nl. Cells were loaded into abeveled glass micropipette (Wiretrol 5 ul, Drummond Scientific Company)mounted on a stereotactic hydraulic injector. P2 CB.17-SCID pups(Charles River) were anesthetized through hypothermia and positioned ina clay head mold on the injector platform, 10-100,000 cells perinjection site were delivered transcranially into the right cerebralcortex using the following coordinates: 0.9 mm from the midline(sagittal sinus), and 2.6 mm from lambda. The depth of injection was0.45 mm from the skin surface. All transplantation experiments adheredto approved UCSF Institutional Animal Care and Use Committee protocols.

Initially, day 35 monolayer cultures were sorted for NKX2.1-GFP+,without PSANCAM selection, and injected into newborn SCID mouse cortex.However, within 4 months, injected mouse brains (12/12 mice) containedtumor-like overgrowths at the injection site and/or at the pial surfacenear the injection tract (data not shown). These were not pluripotentcell-derived teratomas; OCT4+ cells or polarized neuroepithelialrosettes were not found. The growths primarily contained NKX2.1-GFP+neural cells, Tumors were defined as a core of human-specific nuclearantigen (HNA) positive and KI67+ human cells persisting for more than 4months post-injection (MPI). Initially tumor incidence was a surprisebecause day 35 cultures contained seemingly few neural progenitor cellsexpressing KI67, GFAP, or OLIG2 (FIG. 3E). But, focal growths (˜1focus/2500 plated cells, n=3) were occasionally detected in extendedco-cultures, consistent with tumor formation in vivo. Therefore, weperformed protocol optimization to impede these growths. Low-densitymonolayer culture promoted neuronal differentiation and lowered tumorincidence to 50% (6/12 mice). Tumor incidence was further reduced to 33%(3/9 mice) by brief addition of DAPT, a gamma secretase inhibitor ofNotch signaling, to induce neuronal differentiation prior to injection.In vitro focal growths (n=3) and in vivo tumor incidence were eliminated(0/6 mice) when day 35 cultures were pretreated with DAPT andFACS-sorted for both NKX2.1-GFP+ and PSA-NCAM+ cells prior to co-cultureor transplantation, whereas GYP+ and PSA-NCAM-negative cells continuedto form foci and tumors (3/4 mice). A summary of injected animals isprovided (FIG. 16).

Immunostaining

Cultured cells were fixed in 4% paraformaldehyde for 10-20 min. Mousebrains were fixed overnight-4° C. after trans-cardial perfusion andsectioned (50 μm) by vibratome or sliding microtome. EBs and humantissue were sectioned (25 μm) by cryostat, Cells and sections werestained: 5-1.0 min antigen retrieval with boiling 0.01 M citrate bufferpH=6, 1 hr block with 5% serum and 0.1% triton X100 in PBS, overnight-4°C. primary antibody in block buffer (except triton-free buffer used forGABA antibody), wash 3× in PBS+tritonX100, 2 hr secondary antibody(Invitrogen), wash 4× in PBS+tritonX100. Primary antibodies are listedin Table 2,

TABLE 2 Antibodies Antibody Company Catalog# Dilution ASCL1 Cosmo BioSK-T01-003 1-500 CALB Swant CB 38 1-2000 CALR Swant 7699 1-2000 CHATMillipore AB144P 1-300 COUPTFII Perseus Proteomics PP-H7147-00 1-1000DARPP32 Santa Cruz sc-11365 1-1000 DCX Cell Signaling 4604S 1-500 DLX2Gift from K. Yoshikawa 1-1000 ER81 Covance PRB-362C 1-1000 FOXG1 Giftfrom Y. Sasai 1-500 GABA Sigma A2052 1-2000 GFAP Millipore MAB3402 1-500GFP Aves Labs GFP-1020 1-1000 HNA Millipore MAB1281 1-500 ISLET1 DSHB39.4D5 1-200 KI67 Abcam ab15580 1-500 NCadherin BD Biosciences 5615541-50 NEUN Millipore MAB377 1-150 NEUN Millipore MAB377B 1-150 NKX2.1Novacastra TTF-1-L-CE 1-150 NKX2.1 Santa Cruz sc-13040 1-250 NKX2.2 DSHB74.5A5 1-100 OLIG2 Millipore AB9610 1-500 PAX6 Millipore AB5409 1-250 PVSigma P3088 1-4000 PV Swant PV-25 1-2000 RAX Gift from Y. Sasai 1-500RFP Clontech 632496 1-500 RFP Chromotek 5f8 1-1000 SST BachemT-4103.0050 1-500 TBR1 Abcam ab31940 1-500 TH Pelfreeze P40101-0 1-1000TUJ1 Covance MMS-435P 1-1000 VGAT Synaptic Systems 131 003 1-500Transcript Expression

RNA was prepared from cell pellets with RNEasy kit (Qiagen). CDNA wasprepared with Superscript III-first strand kit (Invitrogen),Quantitative RTPCR was performed with SYBR green master mix on areal-time PCR system (Applied Biosystems), Reverse-transcriptasenegative controls were used. Amplicon specificity was determined bygel-electrophoresis and melt-curve analysis. Primer sequences are listedin Table 3.

TABLE 3 Primer Sequences Gene Strand Primer Sequence ASCL1 FGTCCTGTCGCCCACCATCTC ASCL1 R CCCTCCCAACGCCACTGAC CALB2 FTCAGAGATGTCCCGACTCCTG CALB2 R GCCGCTTCTATCCTTGTCGTAA DLX2 FGCCTCAACAACGTCCCTTACT DLX2 R TCACTATCCGAATTTCAGGCTCA FOXG1 FAGAAGAACGGCAAGTACGAGA FOXG1 R TGTTGAGGGACAGATTGTGGC GAD1 FCGAGGACTCTGGACAGTAGAGG GAD1 R GATCTTGAGCCCCAGTTTTCTG GAPDH FGGTGGTCTCCTCTGACTTCAAC GAPDH R TTCGTTGTCATACCAGGAAATG LHX6 FTCTGCAAGATGGACTACTTCAGC LHX6 R CTTGGGTTGACTGTCCTGTTC NKX2.1 FAGACTCGCTCGCTCATTTGT NKX2.1 R CTCCATGCCCACTTTCTTGT NPY FCGCTGCGACACTACATCAAC NPY R CAGGGTCTTCAAGCCGAGTT OLIG2 FAGCTCCTCAAATCGCATCC OLIG2 R ATAGTCGTCGCAGCTTTCG POU5F1 FGCAAAACCCGGAGGAGGAGTC POU5F1 R CCACATCGGCCTGTGTATATC PVALB FAAAGAGTGCGGATGATGTGAAG PVALB R ACCCCAATTTTGCCGTCCC SST FGCTGCTGTCTGAACCCAAC SST R CGTTCTCGGGGTGCCATAG TUBB3 F GCAACTACGTGGGCGACTTUBB3 R CGAGGCACGTACTTGTGAGA

For microarray analysis, RNA was submitted to the Southern CaliforniaGenotyping Consortium for hybridization to lumina Human HT-12 v4.0expression bead-chip. hESC=one sample and three technical replicates.D20=three independent samples. D35=one sample and two technicalreplicates. D55=one sample and one technical replicate. Data wasanalyzed with GenomeStudio (Illumina) software, Probes without signalwere validated by confirming hybridization to control human brainreference RNA and/or to samples archived in ArrayExpress.

Electrophysiology and Optical Methods

The patch electrodes were made from borosilicate glass capillaries(B-120-69-15, Sutter Instruments) with a resistance in the range of 5-7MΩ. The pipettes were tip-filled with internal solution containing (inmM): 125 K-gluconate, 15 KCl, 10 HEPES, 4 MgCl₂, 4 Na₂ATP, 0.3 Na₃GTP,10 Tris-phosphocreatine, 0.2 EGTA.

For cultured neurons, the bath was constantly perfused with freshrecording medium containing (in mM): 145 NaCl, 3 KCl, 3 CaCl2, 2 MgCl2,10 HEPES, 8 glucose. Transverse slices (300 μm) were cut on a tissuechopper (Leica VT1200S) and maintained in an incubation chamber withaCST containing (in mM): 110 Choline Cl, 2.5 KCl, 0.5 CaCl2, 7 MgCl2,1.3 NaH2PO4, 25 NaHCO3, 10 glucose. Slice recording medium contained (inmM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1.3 MgCl2, 1.3 NaH2PO4, 25 NaHCO3, 10glucose. Recordings were made with an Axon 700B patch-clamp amplifierand 1320A interface (Axon Instruments). Signals were filtered at 2 kHzusing amplifier circuitry, sampled at 10 kHz, and analyzed using Clampex10.2 (Axon Instruments).

Photostimulation was delivered by mercury lamp (75 mW) with a GFPexcitation bandpass filter and light pulses were generated by Maste-8(A.M.P.I.) through a high-speed shutter (UNIBLITZ), the power density ofthe blue light (Boyden, E. S., et al. (2005). Nat Neurosci 8, 1263-1268)(Nagel, G., et al. (2003). Proc Natl Acad Sci USA 100, 13940-13945) was8-12 mW·min-2, measured with a power meter (Coherent Instruments),

Statistical Analyses

Data are presented as mean±s.e.m. FIG. 3E data are represented as mean %of co-expressing/GFP+ cells, FIG. 4C-H data as mean bead signalintensity. FIG. 5C data as mean % of UbC-RFP+, ChR2-YFP+, oar TUJ+neurons. FIG. 7D-E data as mean % of HNA+ or UbC-RFP+ human cells.Statistical comparisons used one-way ANOVA with post hoc Bonferroni testfor electrophysiology data, and used a twotailed, two-sample unequalvariance Student's t-Test for immunostaining data. Cell counts werecalculated with Imaris (Bitplane) software using MATLAB plugin. Asummary of sample sizes and cell counts for each experiment and markerare listed in Table 4.

TABLE 4 Summary of sample sizes and cell counts for each figure, marker,and stage of differentiation. Number of Differentiation TotalExperiments/ Positive Cells FIG. Marker Stage Animals Cells Counted 3ENKX2.1 5 wk 6 1103 1219 3E FOXG1 5 wk 6 946 1320 3E NKX2.2 5 wk 5 1381213 3E PAX6 5 wk 2 0 808 3E ASCL1 5 wk 2 476 622 3E COUPTFII 5 wk 2 7848 3E OLIG2 5 wk 2 21 331 3E GFAP 5 wk 3 58 786 3E KI67 5 wk 3 33 10443E TUJ 5 wk 9 1777 2204 3E GABA 5 wk 7 955 1292 3E DLX2 5 wk 2 391 4723E DARPP32 5 wk 2 0 848 3E ER81 5 wk 2 4 954 3E ISLET1 5 wk 4 139 14673E CHAT 5 wk 3 2 2082 3E TH 5 wk 3 64 2082 3E TBR1 5 wk 3 5 1691 5C GFP5 wk 5 314 342 5C GFP 10 wk  3 333 376 5C GFP 20 wk  3 416 517 5C GFP 30wk  3 132 211 5C GABA 5 wk Same as 3E - 5 wk 5C GABA 10 wk  4 801 11135C GABA 20 wk  1 38 56 5C GABA 30 wk  2 140 162 5C VGAT 5 wk 6 553 14245C VGAT 10 wk  2 162 211 5C VGAT 20 wk  3 127 169 5C VGAT 30 wk  1 16 215C CALB 5 wk 5 488 1639Development of the B27+5F Method

We compared three published protocols (methods #1, #2, and #3) for theirability to induce NKX2.1+MGE precursor cells from hESCs. The firstmethod (#1) reported hESC-derived NKX2.1+ and FOXG1+MGE precursor cellsat ˜13% efficiency (Watanabe, K., et al. (2007). Nat Biotechnol 25,681-686). This 35-day protocol utilized serum-free (knockout serumreplacement (KSR)-early/B27-late) supplemented medias and dual-SMADinhibition of BMP (via BMPRIA-Fc) and activin/nodal (via SB431542)signaling pathways to direct neural ectoderm-like differentiation, alongwith WNT pathway inhibition (via DKK1) to specify anteriorforebrain-like identity of embryoid bodies (EBs). From day 24-35,inhibitors were removed, and sonic hedgehog (SHH) was added to specifyventroanterior forebrain-like cells. The second method (#2) reported.NKX2.1+ and FOXG1+MGE precursor cells at 84% efficiency after 28 days inserum-free (KSR/N2) supplemented medias (Li, X. J., et al, (2009).Development 136, 4055-4063). Dual-SMAD inhibition was not used, but SHHtreatment, with or without simultaneous WNT inhibition, was initiatedearlier during differentiation (day 10-28). Although these protocolswere reported to generate FOXG1+ and NKX2.1+ cells, ventraltelencephalic MGE-like versus POA/septum-like identity was notinvestigated.

In our hands, method #1 (FIG. 8B), method #2 (not shown), and anoptimized version of method #1 (FIG. 9A) were unable to generateNKX2.1-GFP+MGE precursor-like cells. The same was true when SHH was usedinstead of purmorphamine. However, we found that a hybrid method couldgenerate NKX2.1-GFP+MGE precursor-like cells at 12% efficiency asquantified by fluorescence activated cell sorting (FACS) (FIG. 5C). This25-day hybrid method involved dual-SMAD (via SB431542 and BMPRIA-Fc) andWNT (via DKK1) inhibition throughout the protocol and simultaneousSmoothened agonist, purmorphamine, treatment from day 10-25 inserum-free (KSR/B27) supplemented medias. A similar efficiency wasachieved in B27/B27 supplemented media (not shown). We assumed that GFP+cell induction resulted from early (d10) Still pathway activation, incontrast to late (d24) SHH from method #1, and we hypothesized that evenearlier addition of purmorphamine (from day 0-25) would increase thepercentage of NKX2.1+MGE precursor cells. However, this modificationresulted in decreased (1.9%) efficiency in KSR/B27 media (FIG. 8D).

However, replacing KSR with B27 supplemented media throughout the 25-dayprotocol, along with early addition of five factors [Rho-associatedprotein kinase (ROCK) inhibitor (Y27632), dual-SMAD inhibitors (SB431542and BMPRIA-Fc), WNT inhibitor (DKK1), and Smoothened agonist(purmorphamine), surprisingly resulted in most cells (70.2%) becomingNKX2.1-GFP+MGE precursor cells (FIG. 8E). Furthermore, when theinhibitors were removed after two weeks of differentiation, and theprotocol extended to day 35, we achieved NKX2.1-GFP+ differentiationefficiencies up to 90.8% by FACS analysis (FIG. 8F). Thus, earlyactivation of the SHH pathway in combination with B27, or lack of KSR,media (B27+5F method) directed efficient ventral forebrain-likedifferentiation from hESCs.

During this study, a third method (method #3) reported to directhypothalamic forebrain-like differentiation from HES-3 NKX2.1GFP/w hESCsat 12-14% efficiency in the presence of FGF2 and retinoic acid(Goulburn, A. L., et al. (2011). Stem Cells 29, 462-473), SHH was notused, but the media appeared to induce SHH, and WNT inhibitor,expression in their cultures and promoted a ventro-anterior neural fatedespite use of retinoic acid, which can act as a caudalizing agent.After 50 days, some cells expressed FOXG1, but the efficiency and fatesof these cells were not determined. In our hands, method #3 generatedNkx2.1-GFP+ cells at ˜7% efficiency. In line with hypothalamic-likeidentity, method #3 generated less NKX2.1+ expression and more NKX2.2+cells on day 24 compared to the B27+5F method (data not shown).

SHH pathway activation was required for NKX2.1-GFP+ cell derivation. Inthis study, we used 1-2 μM of purmorphamine or 500 ng/mL of SHH.Although 2-60 μM of purmorphamine slightly increased GFP+differentiation efficiency in EB cultures, these higher concentrationsdecreased the viability of later-stage monolayer cultures (not shown).We also investigated whether WNT inhibition was dispensable forhESC-telencephalic-like identity, as suggested previously (Li, X. J., etal. (2009). Development 136, 4055-4063). While GFP+ cell differentiationefficiency was similar, DKK1 WNT inhibitor absence resulted in adecrease in the number of cells expressing telencephalic FOXG1 from 70%to 45% (not shown). In addition to WNTs, FGFs act as importantrostra-caudal patterning factors during neural development (Borello, U.,et al. (2008). Neural Dev 3, 17) (Mason, I. (2007). Nat Rev Neurosci 8,583-596) (Ye, W., et al. (1998). Cell 93, 755-766), and FGF8 has beenimplicated in MGE telencephalic-like development from mouse ESCs (Danjo,T., et al. (2011), J Neurosci 31, 1919-1933), We did not add FGF8 to ourcultures, but we did detect FGF8 transcript expression, in agreementwith its role downstream of SHH (Gutin, G., et al. (2006), Development133, 2937-2946) (Ohkubo, Y., et al. (2002). Neuroscience 111, 1-17).Addition of an FGF inhibitor (PD173074) from the onset ofdifferentiation (d0-25) caused a decrease in cells expressing FOXG1(55%), but addition of the same inhibitor on day 14 (d14-25) had noeffect on FOXG1 levels (not shown). Therefore, similar to fetalforebrain development, early inhibition of WNT and activation of FGFsignaling pathways, along with SHH activation, play roles in patterningthe ventral-telencephalic-like identity of hESC-derived cultures.

Cost-effective cell production methods will be preferred over lengthyprotocols and expensive recombinant protein-based reagents, andcryopreservation of cells will facilitate future work. Here, we used 3small molecules (Y27632, SB431542, and purmorphamine) and 2 recombinantproteins (BMPRIA-Fc and DKK1). Several small molecule substitutes nowexist for inhibition of BMP and WNT pathways. We found that dorsomorphin(BMP pathway inhibitor) and CKI-7 (WNT pathway inhibitor) could replacethe proteins in the B27+5F method (Kim, D. S., et al. (2010). Stem CellRev 6, 270-2810) (Osakada, F., et al. (2009). J Cell Sci 122,3169-3179). Although cell viability was reduced by ˜50%, NKX2.1-GFP+efficiency was comparable (not shown). In addition, ˜25% of hESC-derivedMGE-like cells were viable after cryopreservation and thawing, andthawed cells matured into neurons with functional properties asconfirmed by electrophysiology.

For clinical use, GMP-grade hPSC lines may be required. Accordingly, weexamined cGMP-matched hESC lines (ESI17, ESI35, ESI51, and ESI53(Biotime)) for their ability to generate NKX2.1+MGE-like cells. All ofthe lines were similar to HES-3 NKX2.1+ differentiation efficiency usingthe B27+5F method (see Example 8), Since the clinical grade lines do nothave the NKX2.1 knock-in reporter, future work may be needed todetermine acceptable impurity thresholds. Perhaps 75% purity ofhESC-derived NKX2.1+ cells will be sufficient, particularly if theremaining impurities represent non-MGE-type GABAergic interneurons.Suggesting this possibility, we observed that most HES3NKX2.1-GFP-negative neurons expressed GABA, and some expressed TH, butnone expressed TBR1 or CHAT excitatory neuronal markers. To obtain afurther purified composition of the NKX2.1+MGE-like cells antibodies toMGE-specific cell markers, DNA plasmids, RNA, or virus to delivertransgenes (selectable marker, fluorescence, antibiotic resistance, geneoverexpression/inhibition) that select for MGE cells or interneurons(Potter, G. B., et al. (2009). Mol Cell Neurosci 40, 167-186), orFACS-/magnetic MACS-based purification, or anti-mitotic compounds (suchas AraC or MitoC) may be used.

Lentivirus Preparation

Self-inactivating lentiviral plasmids, FUGW-UbC-RFP (RFP dimer2) orpLenti-Synapsin-hChR2(H134R)-EYFP-WPRE (kind gift from Karl Deisseroth),were co-transfected with delta8.9 and VSVG plasmids into 293T cells(ATCC). Lentiviral particle supernatants were collected, concentrated byultracentrifugation, and used to transduce cells overnight with 8 μg/mLpolybrene (Millipore).

Example 1: hESC-Derived Telencephalic MGE-Like Identity

To facilitate the identification of hPSC-derived NKX2.1+MGE precursorcells, we used the HES-3 hESC line (HES-3 NKX2.1^(GFP/w)) with a GFPknock-in construct inserted into the second exon of NKX2.1 (Goulburn, A.L., et al. (2011). Stem Cells 29, 462-473). NKX2.1 expression marksventral forebrain-specific identity in the developing nervous system,including telencephalic MGE, pre-optic area (POA), septum, anddiencephalic hypothalamus. In combination with dual-SMAD (SB431542 andBMPRIA-Fe) and WNT inhibition (DKK1), we found that early SHH pathwayactivation (purmorphamine) and B-27 supplementation enabled highlyefficient and reproducible ventral forebrain-like differentiation fromhESCs. The average NKX2.1-GFP+ efficiency on day 20-30post-differentiation was 74.9±2.1% (n=25 independent differentiationexperiments). We also found that additional hPSC lines, research gradecGMP-matched ESI-17, 35, 51, and 53 (Biotime), and H9 (WiCell), hESClines, and a human adult melanocyte-derived hiPSC line, differentiatedinto NKX2.1+MGE precursor cells at a similar efficiency. The optimizedB27+ five factor (B27+5F) method is outlined in FIG. 1A, FIG. 8. MGEprecursor cells are also referred to as MGE-like progenitors.

To determine the regional identity of the NKX2.1-GFP+ cells, weperformed a 50-day time course of suspension embryoid body (sEB)differentiation and immunostaining analysis for markers of forebraindevelopment (FIGS. 1B and 9). We detected robust NKX2.1-GFP expressionby day 10 of differentiation that colocalized with NKX2.1 protein andwas expressed throughout the time course. In contrast, PAX6, a marker ofdorsal telencephalic neural progenitors, was not detected. Telencephalonmarker, FOXG1, was found in most cells by day 20 and remained highlyexpressed. In opposition to this trend, NKX2.2 expression, a marker ofhypothalamus and more ventrocaudal regions, was primarily only expressedbetween days 10-20. Additional ventral telencephalic progenitor markers,OLIG2 and ASCL1 (MASH1), were induced by day 20-30 (FIG. 9). ISLET1 isstrongly expressed in lateral GE (LGE) and POA, and is expressed inscattered cells within the MGE; it was induced at later (d30-40) timepoints. By 30 days, the cells expressed the migratory neuronal marker,doublecortin (DCX), and the neurotransmitter, GABA. We did not detectthe hypothalamic marker RAX or cholinergic neuron marker CHAT during thetime course. Therefore, the hESCderived NKX2.1-GFP+ cells appeared torepresent a telencephalic MGE-like progenitor and GABAergic neuronallineage. A summary of marker expression during sEB culture is provided(FIG. 15).

FIG. 1. hESC-derived. Telencephalic MGE like interneuron Precursor Cells(also called MGE precursor cells). (A) Outline of B27+5F method used togenerate MGE precursor cells and GABAergic interneurons derivedtherefrom, and corresponding figures. Abbreviations: sEB=suspensionembryoid body; aEB3=adherent embryoid body; ML=monolayer;FACS=fluorescence activated cell sorting; Y27632=Rho-associated kinase(ROCK) inhibitor; SB431542=inhibitor of the TGFβ1 activin receptor-likekinases; BMPRIA=Bone Morphogenetic Protein Receptor 1a Fe chimera;DKK1=Dickkopf homolog I; PM=Purmorphamine; BD F=Brain-derivedNeurotrophic Factor; DAPT=inhibitor of γ-secretase. See also FIG. 8. (B)Sections of sEBs and representative immunofluorescence analysis showingNKX2.1-GFP, PAX6, FOXG1, and NKX2.2 expression. Blue: DAPI. Scale Bar:100 μm. See also FIG. 9 and FIG. 15.

FIG. 8. FACS analysis of differentiated hESCs showing modifications topreviously published protocols and the induction of NKX2.1-GFP+ cells byearly SETH pathway activation combined with B27 media supplementation,related to FIG. 1. (A) Method #1a: KSR-N2-B27 supplemented media alongwith late (d25) PM. (B) Method #1b: KSR-B27 media with late (d25) PMtreatment. (C) Method #1/2 hybrid: KSR-B27 media with mid (d10) PMtreatment. (D) Similar to (C) but with early (d0) PM treatment, (E)Similar to (D) but with B27-B27 media to induce 70% GFP+ efficiency, (F)Similar to (E): B27-B27 media and early (d0) PM addition, but withinhibitor removal on d14 and PM treatment to d35 to induce up to 90%GFP+ efficiency. Tinted histograms=differentiated cultures. Emptyhistograms=undifferentiated culture controls.

FIG. 9. hESC-derived telencephalic MGE-like identity and GABAergicneuronal fate, related to FIG. 1 and FIG. 15. Additional immunostaininganalysis of sEB sections showed induction of the ventral telencephalicmarkers OLIG2 and ASCU (early), ISLET1 (late), and mitotic marker KI67(throughout). The migratory neuronal marker, DCX, and GABA were inducedover time. In contrast, the hypothalamic marker, RAX, and thecholinergic neuronal marker, CHAT, were not detected after 50 days ofdifferentiation. Blue=DAPI.

FIG. 15. A summary of marker expression during suspension embryoid bodydifferentiation from hESCs, related to FIGS. 1 and 9.

Example 2: hESC-Derived MGE Precursor Cells Exhibited VZ and SVZ RadialGlial-Like Stem Cell Behaviors

A defining feature of embryonic neural development is the acquisition ofapico-basal polarity and development of radial glial neural stem cells(Kriegstein and Gotz, 2003). There is evidence that neuroepithelialprogenitors in hESC-derived neural rosettes represent apico-basal-likepolarity (Elkabetz, Y., et al, (2008), Genes Dev 22, 152-165). When sEBswere plated en-bloc on day 4-7, the adherent EBs (aEBs) flattened andrevealed the organization of NKX2.1-GFP+ cells in rosette structures(FIGS. 2A and 2B). N-cadherin expression was restricted to the rosetteluminal surface, confirming polarity, and was consistent withlocalization to radial glial end feet on the apical ventricular surfacein the embryonic brain. KI67 was expressed in many cells, particularlyin those near the rosette lumen. In contrast, neuronal markers, ASCL1and DCX, were detected away from the lumen (FIG. 2B).

Rosettes were labeled with RFP [UbiquitinC promoter-RFP (UbC-RFP) virus]and were monitored by live time-lapse imaging. We detected NKX2.1-GFP+and UbC-RFP+ cells in rosette structures displaying ventricular zone(VZ) radial glia (vRG)-like interkinetic nuclear migration (INM)behavior prior to division (FIGS. 2C and 2E). VRG-like cell bodiestranslocated toward the rosette lumen and divided with a verticalcleavage plane (parallel to the fiber). Interestingly, daughter cellsappeared to extend radial fibers, resembling the symmetrical divisionsattributed to embryonic vRGs that divide with a vertical cleavage plane.

We next investigated whether recently described (Fietz, S. A., et al(2010). Nat Neurosci 13, 690-699) (Hansen, D. V. et al, (2010). Nature464, 554-561) human-enriched outer sub-VZ (SVZ) radial glial (oRG)-likecells were present in our cultures. We focused on NKX2.1-GFP+ cells withunipolar fibers located away from the rosette dusters (FIGS. 2D and 2F).We discovered GFP+ oRG-like cells displaying mitotic somal translocation(MST) prior to division. These cell bodies translocated toward theunipolar fiber and divided with a horizontal cleavage plane(perpendicular to the fiber). In summary, hESC-derived MGE-likeprogenitors (also called MGE precursor cells) could recapitulate VZ andhuman-enriched SVZ radial glial-like stem cell behaviors.

FIG. 2. hESC-MGE-like Progenitors (also referred to as MGE precursorcells) Exhibited VZ and SVZ. Radial Glial Stem Cell-like Divisions, (A)sEBs plated en-bloc on day 4-7, and aEBs fixed on day 14 for analysis;or aEBs were infected with UbC-RFP virus and live cultures time-lapseimaged. (B) Day 14 NKX2.1-GFP expression and a panel of markers in redshown merged and separate: N-Cadherin, KI67, ASCL1, and DCX. Blue: DAPI.Scale Bar: 50 μm. (C) A cluster of rosettes with RFP fluorescence aloneor merged with NKX2.1-GFP. (D) NKX2.1-GFP expressing cells outside ofthe clusters. C, D Scale Bar: 100 μm. (E) Time-lapse imaging series ofboxed region (C) showing three RFP+ cells (blue, orange, and greenarrowheads) that displayed vRG-like INM behavior: translocation towardthe rosette lumen and division (star) with a vertical cleavage plane.Time: hours. Scale Bar: 20 μm. (F) Time-lapse series of boxed region(D). A GFP+ cell with characteristic unipolar morphology (whitearrowhead and smaller arrowheads to mark fiber) exhibited oRG-like MSTbehavior: translocation toward the fiber (46 μm) and division (star)with a horizontal cleavage plane. Time: hours. Scale Bar: 50 μm.

Example 3: hESC-Derived MGE-Like Progenitors Generated GABAergicInterneurons

We further explored the identity and fate of the hESC-derived cultures.Immunostaining analysis was conducted on d25 aEBs (FIGS. 3A and 3B). Day25 aEBs were also dissociated, cultured as a monolayer (ML), andimmunostaining analysis was performed and quantified on day 35 (FIGS.3C-3E). At both day 25 and day 35 stages, NKX2.1-GFP expression wasspecific to cells expressing endogenous NKX2.1 protein (91.3±1.7%) (FIG.3E). On day 25, FOXG1 and OLIG were expressed in most of the NKX2.1-GFP+cells, providing further evidence for ventral telencephalic-likeidentity (FIG. 3B). By day 35, most GFP+ cells continued to expressFOXG1 (81.5±3.6%) but had downregulated OLIG2 (6.8±3%). The majority ofGFP+ cells also expressed ASCL1 (79.9±6.5%) and DLX2 (79.8±3.7%) by day35. Since OLIG2 marks GE progenitors while DLX2 marks GABAergic neuronallineages, these results suggested that day 25 hESC-derived MGE-likeprogenitors (also referred to herein as MGE precursor cells) began todifferentiate into GABAergic neurons by day 35. Indeed, neuronalidentity was confirmed by TUJ1 (92±2.4%) staining (FIGS. 3D and 3E).Most of the GFP+/TUJ1+ cells co-expressed GABA (75.8±2.3%). Theinterneuron subtype marker Calbindin (CALB1 or CALB) was also expressedby day 35 (31.1±5.4%), but other interneuron subtype markers Calretinin(CALB2 or CALR), Somatostatin (SST), and Parvalbumin (PVALB or PV) werenot detected at this stage, except in rare instances for SST or CALR.

A minority of GFP+ cells expressed the diencephalic/oligodendrocytemarker NKX2.2 (13.6±4.7%), neural progenitor/glial cell markers GFAP(3.9±3.9%) and KI67 (2.8±1.5%), LGE/POA-enriched marker ISLET1(7.6±3.3%), or dopaminergic neuron marker TB (4.4±1.3%) (FIG. 3E).Virtually none of the GFP+ cells expressed the neocortical marker PAX6,caudal GE (CGE)/dorsal MGE marker COUPTFII, striatal medium spiny neuronmarker DARPP32, globus pallidus projection neuron marker ER81/ETV1,cholinergic neuron marker CHAT, or glutamatergic neocortical projectionneuron subtype marker TBR1 (FIG. 3E). Based on these results,hESC-derived MGE-like progenitors appeared to have differentiated intopredominantly post-mitotic GABAergic interneurons.

FIG. 3, hESC-MGE-like Progenitors Differentiated into Neurons withProperties of Telencephalic GABAergic Interneurons. (A) aEBs fixed forimmunofluorescence staining on day 25. (B) Day 25 MGE-like progenitorcells expressed NKX2.1-GFP, NKX2.1, FOXG1, OLIG2, ASCL1, and DLX2. Blue:DAPI. Scale Bar: 50 μm. (C) aEBs dissociated, replaced as a ML, andfixed for day 35 immunofluorescence. (D) Day 35 dissociated cellsexpressed NKX2.1-GFP, TUJ1, ASCL1, GABA, and CALB. Blue: DAPI. ScaleBar: 50 μm. (E) Quantification of day 35 immunostaining. The majority ofNKX2.1-GFP+ cells expressed NKX2.1, FOXG-1, ASCL1, TUJ, GABA, and DLX2.Data represented as mean±SEM.

Example 4: hESC-Derived NKX2.1-GFP+ Microarray Profiling

We performed microarray profiling to obtain a global transcriptomecomparison of undifferentiated hESCs and FACS-sorted NKX2.1-GFP+populations over a 55-day time course of differentiation: from 20-dayaEBs (blue bars) or 35-day adherent monolayers (orange bars), or from55-day cultures (green bars) that had been previously labeled withUbC-RFP virus, sorted for GFP on day 35, and co-cultured with glialcells (FIG. 4A). The percentage of NKX2.1-GFP+ cells remained at a highlevel in dissociated monolayer culture (˜81% on d35) or in co-culture(˜94% of RFP+ cells on d55) (FIG. 4B). Dendrogram clustering analysisshowed the differentiated GFP+ populations to be more closely related toeach other than to undifferentiated hESCs (FIG. 10A). To inspect theidentities of the hESC-derived GFP+ populations in greater detail, weselected panels of lineage-specific markers and assessed transcripthybridization intensities over the time course (FIGS. 4C-4H and10C-10F). For a subset of markers, quantitative RTPCR was performed andconfirmed the array data (FIG. 10B). Markers of pluripotency were onlydetected in undifferentiated hESCs, whereas markers of a neural lineage(HES5, DCX, SYP, SYN1) were induced in differentiating GFP+ cells.Markers of glial cells, neural crest, or microglia were not detected. Incontrast, GFP+ cells expressed neuronal markers (TUBB3, DCX, SYP, SYN1),and transcript levels increased over time. We then examinedanterior-posterior central nervous system (CNS) patterning and detectedexpression of anterior CNS markers (FOXG1, SIX3, OTX2), In agreementwith prior results, diencephalic (and more caudal) NKX2.2 wastransiently expressed and then downregulated. Unexpectedly, FOXA2, amarker of CNS floor plate, was expressed in day 20 progenitors but wasnot detected at later time points.

We next investigated markers that identify sub-regions of the forebrain.Aside from temporary expression of NKX2.2, markers of diencephalichypothalamus were not robustly expressed (FIG. 10C). Also, dorsalexcitatory neuronal lineage markers were not detected (FIG. 10D).Instead, ventral telencephalic GABAergic neuronal lineage markers(ASCL1, DLX1, DLX5) were expressed along with MGE markers (NKX2.1, LHX6,LHX8, CXCR7), and their expression intensities generally increased overtime (FIGS. 4F and 4H). Markers of non-MGE ventral telencephalon werenot detected, nor were markers of NKX2.1+ POA/septum or globus pallidus(FIG. 10E). GABAergic markers (GAD1, SLC32A1, SLC6A1) were found, butglutamatergic or cholinergic neuronal markers were not expressed (FIG.40). We detected dopaminergic neuronal transcript (TH) but did notidentify many neurons expressing TH protein (FIG. 3E). These resultssuggested that GFP+ cells were of a principally MGE-like GABAergicneuronal lineage.

In addition to cortical GABAergic interneurons, the MGE generatesstriatal GABAergic interneurons as well as GABAergic projection neuronsof the globus pallidus. Since GFP+ cells did not express globus pallidusmarker transcript or protein, we further assessed markers ofcortical/striatal interneuron lineages.

Striatal interneurons maintain NKX2.1 and LHX8 expression, whereasmigratory cortical interneurons extinguish these markers and expressZEB2, MAF, ARX, CXCR7, and CXCR4. In support of a cortical-likeinterneuron lineage, robust CXCR7 expression was detected in GFP+ cells.Although increased ZEB2, ARX, and CXCR4 transcript signals were found atlater stages, overall levels were modest, and NKX2.1 and LHX8 continuedto be expressed, suggesting a striatal interneuron-like lineage and/or acortical-like lineage at an immature stage (FIGS. 4F and 4H). Lastly, ofthe neuropeptide and calcium binding proteins that mark subtypes ofinterneurons, only SST transcript was robustly detected by day 55 (FIG.4H), In summary, the hESC-derived NKX2.1-GFP+ populations representedMGE-like neural progenitor cells and GABAergic cortical- and/orstriatial-like interneurons.

FIG. 4. Microarray Gene Expression Profiling of hESC-MGE-likeNKX2.1-GFP+ Cell Populations. (A) Schematic and legend for microarraydata. Undifferentiated hESCs (black); and FACS-sorted GFP+ cells fromday 20 aEBs (blue), day 35 ML cultures (orange), and GFP+ cells from d35co-cultured to day 55 (green). (B) Representative FACS histogramanalysis of each differentiation stage and undifferentiated hESCcontrols (black). (C-H) Average transcript hybridization signalintensities for marker panels. IN=interneuron, DA=dopaminergic,ACh=cholinergic, Glu=glutamatergic. Data, represented as mean SEM. Seealso FIG. 10.

FIG. 10. Transcript expression profiling of hESC-derived NKX2. GFP+cells, related to FIG. 4.

-   (A) Dendrogram clustering analysis of microarray data identified the    d20, d35, and d55 GFP+ populations to be more closely related to    each other than to the undifferentiated hESCs.-   (B) Quantitative RTPCR analysis of undifferentiated hESCs (black    bars) and 3-week GFP+ cells (blue bars) relative to GAPDH    expression. Data represented as mean±SD. (C-F) Additional markers    from microarray analysis. Legend: undifferentiated hESC (black), d20    GFP+ (blue), d35 GFP+ (orange), and d55 GFP+ (green) samples. Panels    show: hypothalamic (C), cortical excitatory neuronal lineage (D),    ventral telencephalic (E), and general fetal developmental markers    (F). GP=globus pallidus, POA=pre-optic area, Sep=septum,    PN=projection neuron. Hypothalamic and cortical excitatory neuronal    markers were not detected. The POA/Sep, GP, and MGE-derived PN    markers, ETV1 and GBX2, were also not detected. The dorsal MGE and    CGE marker, NR2F2 (COUPTFII), was identified at a low level,    consistent with rare GFP+ cells expressing COUPTFII protein (FIG.    3E), and NKX6-2 was also weakly detected at d20. The early embryonic    markers, DPPA4, LIN28, and LIN28B, have been used to estimate the    developmental stage of neural cells derived from human pluripotent    stem cells (Patterson, M., et al. (2012). Cell Res 22, 178-193). In    human fetal spinal cord, LIN28 expression is downregulated by 7gw,    whereas DPPA4 and LIN28B are not reduced until 13gw. In our    cultures, DPPA4 and LIN28 were not detected by d35, and LIN28B    expression persisted to d55. These results suggest d35-55 GFP+ cells    may be similar to a 7-13gw fetal developmental stage. Data    represented as mean±SEM.

Example 5: Protracted Maturation of hESC-Derived MGE-Like Cells intoSubtypes of GABAergic Interneurons

We next sought to more convincingly determine both the neuronal subtypesgenerated by the NKX2.1-GFP+MGE-precursor like cells and thedevelopmental timeline of subtype maturation, To study their maturation,day 35 FACS-sorted GFP+ cells were co-cultured with cortical glial cells(FIG. 5A), and some cells were labeled with UbC-RFP virus prior toco-culture. Cultures were fixed for analysis after five, 10, 20, and 30weeks post-differentiation (WPD), and neuronal subtype marker expressionwas quantified (FIG. 5C). Following 30 WPD, RFP+hESC-derived neuronswere notably larger in somal size and expressed the GABAergicneuron-specific marker, VGAT, and interneuron subtype markers SST, CALB,and CALR (FIG. 5B). Images from 10-20-WPD cultures are shown in FIG. 11,Virtually all neurons at five WPD expressed NKX2.1-GFP, but, similar tocortical interneurons, the percentage of NKX2.1+ neurons significantlydeclined by 30 WPD (66.7±6.1%; p=0.03). Most of the neurons expressedGABA (75-86%) and VGAT (53-78%) from 5-30 WPD. Aside from rare cells (11of 3,110 neurons), the excitatory neuronal marker TBR1 was notexpressed. CALB was expressed in neurons throughout the time course(24-36%). In contrast, the percentage of SST+ and CALR+ neuronsincreased over time and were significantly induced from 10 to 20 WPD(SST: 2.8±1% to 12.8±9%; p=0.03, and CALR: 8.8±4.9% to 52.6±6.2%;p=0.004). By 30 WPD, the percentage of SST+ neurons increased to40.6±8.6%, and CALR+ neurons increased to 77.7±14.9%. Conversely, PV+neurons were not detected by 30 WPD (0 of 1,146 neurons), and NPY+neurons were rare (6 of 819 neurons, not shown). Thus, NKX2.1-GFP+MGE-like cells matured into NKX2.1+ and NKX2.1-negative GABAergicinterneurons expressing CALB, CALR and/or SST subtype markers, andpronounced SST and CALR subtype maturation occurred between 10 and 20WPD.

It was surprising that our hESC-derived GABAergic interneurons required20-30 weeks to show substantial expression of SST and CALR. However,this protracted timeline of differentiation is similar to human fetaland infant interneuron subtype development (Fertuzinhos, S., et al,(2009), Cereb Cortex 19, 2196-2207). We confirmed these findings withour own histological analysis of developing human cortex and MGE (FIGS.12A and 12B), To further investigate human fetal MGE-derived fates, wedissected, labeled with UbC-RFP virus, and co-cultured 18gestational-week (gw) human fetal MGE cells. By 12 weeks in culture,RFP+ human fetal MGE cells had matured into CALB+, CALR+, SST+, andGABA+ neurons, and they did not express TBR1 (FIG. 12C), Therefore,hESC-derived MGE-like cell maturation paralleled both endogenous andcultured human fetal MGE development: comparable interneuron subtypeswere generated in a similar sequence and time frame.

FIG. 5. hESC-MGE-precursor like Cell-derived GABAergic interneuron

Maturation and Firing Properties.

-   (A) Dissociated ML cultures infected with UbC-RFP lentivirus,    FACS-sorted on day 35 for GFP+ cells, and co-cultured.-   (B) Immunostaining of 30-week cultures showing highly branched RFP+    human neurons that expressed VGAT, SST, CALB, and CALR. Scale Bar:    50 μm.-   (C) Quantification of immunostaining analyses over 5, 10, 20, and 30    weeks. Data represented as mean±SEM. See also FIGS. 11 and 12.-   (D) DIC image of hESC-derived neurons at 12 and 30 WPD, insets show    RFP expression of recorded neurons. Scale bar: 20 μm.-   (E) Statistical results showing membrane resistance (Rm), resting    membrane potential (RMP), membrane capacitance (Cm), and action    potential (AP) ½-width.-   (F) Representative AP firing patterns at each stage upon near    threshold (upper) and superthreshold (lower) current injection.    Scale bars: 50 my and 100 ms. See also FIG. 13.

(G) Average first AP traces upon threshold current injection. Scalebars: 25 mV and 25 ms.

-   (H) Statistical results showing AHPs at each stage (dashed    line=baseline). (I-J) I-V curve of Na⁺(I) and K⁺ currents (J) at    each stage, measured under stepped voltages (500 ms duration), E, H,    I, J: Data represented as mean±SEM. *** represents p<0.001.

FIG. 11. Maturation of hESC-derived MGE precursor-like cells intoGABAergic interneuron subtypes, related to FIG. 5.

Immunostaining analysis of NKX2.1-GFP+ cells pre-labeled with UbC-RFPvirus, FACS-sorted for GFP on d35, and co-cultured for 10 and 20 weekspostdifferentiation (WPD). By 10 WPD, hESC-derived neurons expressedVGAT and Calbindin (CALB1), and rare cells expressed Calretinin (CALB2)or SST. By 20 WPD, human neurons expressed VGAT, CALB1, CALB2, and SST.Parvalbumin (PVALB) was not detected at either time point. Blue=DAPI,

FIG. 12. Development of interneuron subtypes in human fetal cortex andMGE, and in cultures derived from human fetal MGE, related to FIG. 5.

-   (A) Interneuron subtype marker expression in human fetal cortical    sections from 14 15gw, 24gw, and 8mo post-natal. (pn). CALB and CALR    were expressed in all samples, but SST and PV were expressed in 24gw    and 8mo, and 8mo only samples, respectively. Blue=DAPI.-   (B) Subtype marker expression in 15gw human fetal MGE sections. Both    CALB and CALR were expressed and co-localized with NKX2.1.    Blue=DAPI.-   (C) Human fetal MGE was dissociated, labeled with UbC-RFP,    FACS-sorted for RFP+ cells, and co-cultured with dissociated human    fetal cortical cells. RFP+ cells expressed CALB, CALR, SST, and    GABA, but did not express TBR1. Blue=DAPI.

Example 6: Functional Maturation of hESC-Derived MGE-LikeInterneuron-Like Firing Properties, Synapse Formation, and GABAergicOutput

To test whether the hESC-derived cells were functional neurons, weperformed whole-cell patch recordings to examine theirelectrophysiological properties at different WPD (8 weeks, n=21; 12weeks, n=35; 15 weeks, n=31; 30 weeks, n=18). We found that actionpotential (AP) firing patterns of hESC-derived neurons were quiteimmature at eight WPD, judged by the broad AP ½-width of the first AP,small after-hyperpolarization (AHP), and inability to fire repetitivelyupon high current injection (FIGS. 5E-5H). The peak voltage-gated Na+and K+ channel currents increased significantly from eight to 12 WPD(FIGS. 5I and 5J), concomitant with a significant decrease in membraneresistance (Rm) (FIG. 5E). Many neurons showed more mature repetitive APfiring upon near threshold current injection at 12 and 15 WPD (FIGS. 5F,13B and 13C). By 30 WPD, hESCderived neurons exhibited high-frequencyrepetitive AP firing upon superthreshold current injection (FIGS. 5F and13D), along with a corresponding increase in membrane capacitance (Cm)and more hyperpolarized resting membrane potential (RMP) (FIG. 5E). Inaddition, 30-WPD neurons exhibited smaller AP ½-width (FIG. 5E) andlarger AHPs (FIGS. 5G and 5H). Consistent with these more maturebiophysical properties, we also noted more mature morphologies of thehESC-derived neurons with multiple long processes at 30 WPD compared tothe earlier 12 WPD stage (FIG. 5D).

Next, we investigated whether the MGE-like cells were GABAergic neuronsby studying their synaptic properties in co-culture with mouse glialcells. hESC-derived NKX2.1-GFP+ neuronal processes co-localized withpunctate pre-synapatic VGAT expression, suggesting the formation ofGABAergic synapses (FIG. 6A). Spontaneous post-synaptic currents (sPSC)were detected by eight weeks postdifferentiation and were fully blockedby the GABAA receptor inhibitor bicuculline methiodide (BMI, 20 μM),indicating functional GABAergic-specific synapse formation (FIG. 6B).The percentage of neurons receiving sPSCs increased from 33.3% at eightWPD (n=12) to 82.1% at 12 WPD (n=28, FIG. 6C). To confirm that GABAergicneurons were able to send outputs to neighboring neurons, we transfectedhalf of the neurons with Synapsin promoter—Channelrhodopsin2-EYFP(ChR2-EYFP) by lentiviral infection. Blue light stimulation reliablyinduced action potential firing in EYFP-positive neurons (FIG. 13F)(Weick, J. P., et al. (2011), Proc Natl Acad Sci USA 108, 20189-20194),and evoked robust post-synaptic currents (PSCs) in neighboring neurons(FIGS. 6D and 6E). In addition, the PSCs showed a long decay time(31.4±1.9 ms, n=26), characteristic of GABAergic PSCs. This was furtherverified by reversible blockade of light-evoked PSCs by BMI (FIGS. 6Dand 6E). The reversal potential of light-evoked PSCs was −32.7 mV (FIGS.6F and 6G), close to the expected C1-reversal potential under ourrecording conditions [−37.3 mV=−53.4 mV (by Nernst equation)+16.1 mV(junction potential)]. These results suggested that hESC-derivedMGE-like interneurons produced exclusively GABAergic synaptic output.

To examine whether hESC-derived interneurons could form synapses ontoprimary human neurons, MGE-like cells were labeled at four WPD withChR2-YFP and UbC-RFP virus, and RFP+ FACS-sorted cells were co-culturedfor seven weeks with dissociated human fetal cortical cells from 20gw.Whole-cell recordings were obtained from RFP-negative primary corticalneurons after co-culture (FIG. 6H). Blue light stimulation ofhESC-derived neurons induced GABAergic-specific PSCs in recorded primaryneurons that were completely blocked by BMI (FIGS. 6I and 6J).Furthermore, we found polysynaptic responses upon light stimulation(FIG. 6I), which were also blocked by BMI, indicating robust synapticintegration of hESCderived neurons into cultured human fetal neuronalcircuits. Thus, hESC-derived neurons demonstrated functional neuronalproperties, GABAergic-exclusive synaptic output, and slow 30-weekmaturation of interneuron firing properties, consistent with the slowpace of subtype marker expression.

FIG. 6. GARAergic Synaptic Properties of hESC-derived Interneurons.

-   (A) Images showing VGAT expression in hESC-derived NKX2.1-GFP+    neurons at 12 WPD. Right: zoom of dashed rectangle. Scale bar: left,    50 μm; right, 10 μm.-   (B) Traces showing spontaneous post-synaptic currents (PSCs) are    hESC-derived neurons, bottom: PSCs were fully blocked by BMI. Scale    bar: 100 pA, 5 s and 0.25 s (dashed line) for middle trace.-   (C) Percentage of neurons showing spontaneous PSCs at different    stages.-   (D) hESC-derived neurons were transfected with ChR2-EYFP. Traces    show pulses of blue light (blue bar) evoked PSCs in neighboring    cells that were reversibly blocked by BMI, Scale bar: 50 pA and 50    ms, See also FIG. 13.-   (F) Average amplitudes of light-evoked GABAergic PSCs and    application of BMI. (F-G) Traces showing light-evoked (blue bar)    PSCs at different holding potentials. Summarized results (n=7)    showing I-V curve of light-evoked GABAergic PSCs (G),-   (H) Merged image showing DIC of human fetal cortical cells    co-cultured with sorted UbC-RFP+ and ChR2 transfected hESC-derived    neurons. Scale bar: 20 μm.-   (I) Traces showing blue light (blue bar) stimulation of hESC-derived    neuron-evoked PSCs in RFP-negative recorded human fetal cortical    neurons. Upper panel shows PSC mono-synaptic response, lower panel    shows PSC with poly-synaptic responses both fully blocked by BMI.    Scale bar: 50 pA and 50 ms,-   (J) Averaged amplitudes of light-evoked PSCs and application of BMI.    E, J: Data represented as mean±SEM.

FIG. 13. Maturation of hESC-derived interneuron firing properties,related to FIGS. 5 and 6.

-   (A-D) Example traces of action potentials (APs) at different stages    post-differentiation. Stepped currents were injected into recorded    neurons at V_(hold) of −60 mV to −70 mV at different stages: (A) 8    WPD (B) 12 WPD (C) 15 WPD (D) 30 WPD. Red traces indicated APs upon    threshold current injection. Black traces indicated APs upon two or    three-fold times threshold current injection. Scale bars: 50 mV and    200 ms. (E-F) Blue light induced APs in ChR2-EYFP positive    hESC-derived neurons. Merged image of EYFP fluorescence and DIC at    10 WPD (E). Scale bar: 20 μm. Pulses of blue light (blue trace)    reliably induced APs (black trace) in ChR2-expressing neurons (F).

Example 7: hESC-Derived MGE-Like Interneuron Maturation and FunctionalIntegration in the Mouse Brain

To rigorously evaluate cell fate and function, hESC-derived MGE-likecells were transplanted into the mouse brain. We modified our protocolto avoid injection of undifferentiated NI 2.1+ neural stem cells (FIG.16), Treatment with DAPT, a gamma secretase inhibitor of the Notchsignaling pathway, was used to induce neuronal differentiation combinedPSA-NCAM purification of neuronal precursors (Schmandt, T., et al.(2005). Stem Cells Dev 14, 55-64). An average of 75.7±5.2% (n=12) ofNKX2.1-GFP+ cells were positive for high PSA-NCAM expression by FACS(FIG. 14A) NKX2.1-GFP+ and PSA-NCAM+ cells from day 35, enriched forGABAergic neuronal precursors (FIG. 3E), were injected into severecombined immuno-deficient (SCID) newborn mouse cortex (FIG. 7A). Thehuman-specific nuclear antigen (HNA) positive human cells survived forseven months post-injection (MPI) (the longest time point), and somehuman cells migrated more than 1 mm from the injection site (FIGS. 7B,14B and 14C). Human cell survival rates of injected cells) after two,four, and seven MPI were 5.6±2.6%, 3.1±1.5%, and 8.6±3.1%, respectively.After two MPI, human cells expressing HNA and NKX2.1-GFP (67.8±1.6%),KI67 (25.5±1.7%), or DCX (79.8±3.8%) were mostly still located at theinjection site (FIGS. 7B and 14B). But by four MPI, KI67 expression wassignificantly reduced (1.7±0.27%; p=0.04), and DCX expression wassimilarly reduced over time (5.9±4.9% by 7 MPI; p=0.008). Also,NKX2.1-GFP was detected in only 35.6±14% of human cells after sevenMPI—a lower percentage than was found in 30 WPD co-cultures. A reversetrend was found for the post-mitotic neuronal marker, NEUN, whichincreased to 68.4±8.3% of human cells by seven MPI. In contrast, glialcell markers, GFAP and OLIG2, were expressed by a lower percentage ofhuman cells at seven MPI (11.2±4.3% and 10.7±4.4%, respectively). SomehESC-derived cultures were labeled with UbC-RFP virus pre-injection.Following seven MPI in the mouse brain, RFP+ human cells with neuronalmorphologies were found to express GABA, SST, CALB, and CALR (FIGS. 7Cand 7E). PV+ human cells were not detected, except for rare cells withweak signal (4 of 1,829 cells). In summary, MGE-like GABAergic neuronalprecursors injected into the mouse cortex primarily matured into neuronsthat expressed SST, CALR, and CALB interneuron subtype markers.

To examine whether hESC-derived MGE-like cells could develop intofunctional interneurons that synaptically integrate in vivo, weperformed whole-cell recordings of RFP+ human cells in mouse brainslices seven MPI. Intracellular filling with neurobiotin andpost-staining revealed the extensive process branching of recorded RFP+neurons (FIG. 7F). Among 17 total human cells patched from threeanimals, 16 neurons exhibited the ability to fire action potentials withan average RMP of −64.8±4.0 mV. In addition, two groups of interneuronswere identified, type I and type II, with different membrane propertiesand firing patterns. Type I interneurons had an average RMP of −67.3±2.9my, Rm of 257±78 MΩ, and Cm of 69.4±0.6 pF. The firing pattern of type Iinterneurons displayed a significant delay to spike at threshold, andlittle adaptation upon superthreshold, current injection (FIGS. 7G and14D). Type II interneurons had more hyperpolarized RMP (−80.1±3.4 mV),smaller Rm (91±28 MΩ), and smaller Cm (27.75±4.6 pF). The firing patternof type II interneurons showed rapid adaptation of initial spikes uponsuperthreshold current injection (FIGS. 7G and 14E). Furthermore, thetransplanted hESC-derived interneurons received synaptic inputs (16 of16) containing both BMI sensitive GABAergic and6-Cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) sensitive glutamatergiccomponents (FIG. 7H), suggesting functional integration into the hostcortex.

FIG. 7. hESC-derived MGE-like Interneuron Precursor Cell Maturation andFunctional Integration in the Mouse Brain.

-   (A) Day 35 ML cultures FACS-sorted for NKX2.1-GFP and PSA-NCAM, and    injected into newborn mouse cortex. See also FIG. 16.-   (B) Mouse brain tissue sections at 2 and 7 MPI stained for    human-specific hNA, GFP, and KI67. Blue: DAPI. Scale Bar: 200 μm.    See also FIG. 14.-   (C) Histological analysis of human cells labeled with UbC-RFP that    co-expressed (arrow) NEUN, GABA, SST, CALB, and CALR at 7 MPI. Blue:    DAPI. Scale Bar: 50 μm.-   (D-E) Quantification of histology at 2 (black), 4 (orange), and 7    (blue) MPI, and of SST, CALB, and CALR (E). Data represented as    mean±SEM.-   (F) hESC-derived neuron labeled by intracellular filling of    neurobiotin (NB, green). Inset: RFP fluorescence of filled neuron 7    MPI. Scale bar: 20 μm; inset 5 μm.-   (G) Traces of AP firing patterns of type I (left) and type II    (right) hESC-derived neurons upon near threshold (top) and    superthreshold (bottom) current injection at 7 MPI. Scale bars: 50    mV and 100 ms.-   (H) Left panel: traces of spontaneous PSCs recorded from    hESC-derived neurons post-injection; upper right: BMI blocked PSCs    with slow decay-time (arrow), and the remaining PSCs with fast    decay-time (arrow head) were blocked by subsequent application of    CNQX (lower right panel). Scale bars: 50 pA, 2.5 s and 0.2 s (dashed    line) for zoomed traces. See also FIG. 14.

FIG. 14, Maturation of hESC-derived MGE-like interneurons and subtypefiring properties in the mouse brain, related to FIG. 7 and FIG. 16.

-   (A) FACS analysis histogram on d35 showing high expression of    PSA-NCAM by most GFP+ cells (red) compared to isotype antibody    control (grey).-   (B) Immunostaining analysis of migration and maturation of    NKX2.1-GFP+ and PSANCAM+hESC-derived MGE-like cells 2, 4, and 7    months post-injection (MPI). By 7 MPI, human-specific nuclear    antigen (HNA)+ human cells could migrate, downregulate GFP and DCX,    and upregulate NEUN, a marker of neuronal maturation. Blue=DAPI.-   (C) hESC-derived MGE-like cell migration in 6 mice. Human cells were    counted in rostral and caudal cortical sections flanking a single    injection site at 2, 4, and 7 MPI. Some migration was detected by 7    MPI. Plotted as the percentage of injected cells. (D-G) Firing    properties of type I and type II hESC-derived interneurons at 7 MPI.    AP firing patterns upon near threshold (top) and superthreshold    (400-500 pA, bottom) current injection of type I (D) and type II (E)    neurons. Each column (top trace and bottom trace) represents AP    firing patterns of one neuron. Top panels: red trace represents    threshold AP firing pattern; black trace is 2-fold threshold AP    firing pattern. Scale bars: 50 mV and 200 ms. (F) Statistical    results showing the differences in AP characteristics between type I    and type II neurons. Data represented as mean±SEM, and students'    t-test was used for statistical comparisons. * represents    p<0.05. (G) Analysis of AP firing frequency upon superthreshold    current injection. The type II neurons exhibited rapid adaptation    firing properties.

FIG. 16. A summary of hESC differentiation protocol optimization, animaltransplantation, and tumor incidence, related to FIGS. 7 and 14.

Example 8: hESC-Derived MGE-Precursor Like Cells

Clinical grade GMP-matched hESC lines ESI17 (FIG. 17), ESI35 (FIG. 18),ESI51 (FIG. 19), and H9 (FIG. 20) were differentiated into MGE precursorlike cells (top row) and further into interneurons (bottom row).

FIGS. 17-20, Top Row: hESC lines were differentiated with the B27+5Fmethod as suspension embryoid bodies (sEB's) to day 7 followed byadherent EB (aEB) culture to day 28. Cultures were fixed forimmunofluorescence staining, Most of the cells in the adherent EBsexhibited high expression of markers of MGE (NKX2.1), telencephalon(FOXG1), and neuronal specification (ASCL1), The minority of cellsexpressed markers of ventral hypothalamus (NKX2.2) and oligodendrocyteprogenitor cells (OLIG2).

FIGS. 17-20, Bottom Row: Day 28 aEB cultures were dissociated to singlecells, replaced as a monolayer, cultured for an additional 2 weeks inneurobasal media with B27 supplement with or without BDNF, DAFT, SHH andfixed for similar analysis. These day 42 monolayer cultures expressedNKX2.1, neuronal marker (TUJ), and began to express inhibitory neuronmarker (GABA). OLIG2 expression was not detected, and NKX2.2 was onlyfound in rare cells.

Example 9: Derivation of MGE Precursor Cells from Naïve HumanPluripotent Stem Cells

Typical primed HES3 (NKX2.1-GFP) hESCs express homogeneous OCT4 but donot express naïve stem cell marker TFE3 homogeneously in the nucleus(FIG. 21, top row). Primed HES3 (NKX2.1-GFP) hESCs were converted intonaïve HES3 stem cells using published methods (Gafni and Hanna et al,Nature 2013), Naïve hESCs expressed TFE3 in the nucleus of virtuallyevery cell (FIG. 21, middle row). Differentiation of naïve stem cellsusing the B27+5F method resulted in differentiation of the HES3 hESCsinto cells that expressed MGE markers NKX2.1-GFP, NKX2.1, and LHX6 by 2to 6 weeks of adherent EB culture (FIG. 21, bottom row).

Naïve ES cells are more undifferentiated than traditional human ES/iPScells grown in typical media with bFGF only. Traditional hPSCs are moreequivalent to the later-stage post-implantation embryo epiblast thanmouse mPSCs, which are more similar to the earlier-stagepre-implantation inner cell mass. Naïve human ES and iPS cells aretherefore more similar to mouse ES cells. Their properties (geneexpression and epigenetics) are equivalent to the pre-implantationembryo. They can be identified by the expression and nuclearlocalization of the transcription factor TFE3, and by colony morphology.These properties distinguish nave cells from traditional primed hPSCs.

Example 10: Utilization of an MGE-Enriched Enhancer Sequence for theSelection and Purification of Interneurons Derived from MGE PrecursorCells Differentiated In Vitro from hPSC

Transgenic mice that contain various enhancer-reporter transgenes areavailable.

In these mice, the reporter gene is expressed with different patternsand lineage specificities in the forebrain depending on the DNA sequenceof the enhancer (FIG. 22A-D).

Based on their expression pattern in the forebrain of the transgenicmice, MGE-enriched enhancer sequences were cloned into viral vectors todrive the expression of fluorescent reporter genes and/or antibioticresistance genes in an MGE-selective manner. The constructs alsocontained a Rex1-antibiotic resistance cassette to enable the selectionand expansion of stable transgenic hPSCs, (FIG. 22, E).

The intergenic DLX1/2 i12b (422) enhancer driving the mCherry RFPreporter gene (i12b-RFP) was delivered into the HES3 NKX2.1-GFP hESCline using lentivirus, and two stable cell lines were generated (#5 and#10) as confirmed by genomic DNA PCR for mCherry. (FIG. 22, F).

The modified lines were differentiated using the B27±5F method, andi12b-RFP expression was detected after three weeks of differentiation,along with NKX2.1. (FIG. 22, G). i12b-RFP+ cells in EBs co-expressedGABAergic neuron marker DLX2 and neuronal marker TUJ1. (FIGS. 22, H andI).

Flow cytometry analysis confirmed that many cells derived with theB27+5F method co-expressed both NKX2.1-GFP and i12b-RFP (FIG. 22, J).Treatment of these cultures with NOTCH pathway inhibitor, DAPT, resultedin a marked increase in this double positive population of MGE derivedinterneurons (FIG. 22, K).

Double positive (NKX2.1-GFP+ and i12b-RFP+) MGE precursor cells werepurified by FACS and transplanted into the SCID mouse cortex. Severalmonths post-injection, human cells expressing both NKX2.1-GFP andi12b-RFP were found to disperse from the injection site and to integrateinto the surrounding rodent grey matter, consistent with hallmarkproperties of differentiation into MGE derived interneurons. (FIG. 22,L).

Cultured hPSC-derived MGE derived interneurons expressing i12b-RFP (FIG.22, M) were also analyzed using electrophysiology. Recorded RFP+interneurons fired repetitive trains of action potentials, confirmingtheir neuronal fate (FIG. 22, N).

Example 11: Generation of MGE Derived Interneurons Using Long-TermSuspension Culture

HES3 hESCs differentiated using B27+5F conditions as sEB's in normoxicgas (20% oxygen tension) produced MGE interneurons expressing NKX2.1-GFPand LHX6. The cells were analyzed on day 35 of culture (FIG. 23, row A)

HES3 hESCs differentiated using B27+5F conditions as sEBs in normoxicgas produced MGE interneurons expressing NKX2.1-GFP and LHX6. In thisdifferentiation protocol, SHI agonist (purmorphamine) was removed on day21 (in contrast to FIG. 23, row A, above, where SHH agonist(purmorphamine) was present throughout the culture period), The cellswere analyzed on day 35 of culture (FIG. 23, row B).

HES3 hESCs were cultured in GMEM and DMEM/F12 media with KSR, N2, andB27 supplements (added sequentially) and ROCK, WNT, and SMAD inhibitorsand SHH agonist in hyperoxic gas (40% oxygen tension). The cells wereanalyzed on day 35 of culture, (FIG. 23, row C).

HES3 hESCs were cultured in GMEM and DMEM/F12 media with KSR, N2, andB27 supplements (added sequentially) and ROCK, WNT, and SMAD inhibitorsand agonist with matrigel added to the media (1-2%). sEBs weremaintained for more than 50 days in culture in hyperoxic gas. The cellswere analyzed on day 60 of culture. (FIG. 23, row 1)).

Example 12: Generation of MGE Precursor Cells Using Small MoleculeInhibitors of BMP and WNT Signaling Pathways

The following are further examples of small molecule inhibitors of BMPand WNT signaling pathways that are useful for generation of MGEprecursor cells. The differentiation protocol was as described inExample 1.

FIG. 24, A: B27+5F differentiation protocol with BMPR1A and DKK1 asinhibitors of BMP and WNT signaling pathways, respectively, induceddifferentiation of hESCs into MGE precursor cells that co-expressNKX2.1GFP (top) and FOXG1 (bottom). (see also, Example 1 for details).

FIG. 24, B: B27+5F differentiation protocol with LDN193189 (0.1 μM,Catalog #04-0019 (Stemgent)) and XAV939 (2 μM, Catalog #3748 (Tocris))as inhibitors of BMP and WNT signaling pathways, respectively, induceddifferentiation of hESCs into MGE precursor cells that co-expressNKX2.1GFP (top) and FOXG1 (bottom).

FIG. 24, C: B27+5F differentiation protocol with LDN193189 (0.1 μM,Catalog #04-0019 (Stemgent)) and IWIR1e (3 Cayman Chemical Catalog#13659) as inhibitors of BMP and WNT signaling pathways, respectively,induced differentiation of hESCs into MGE precursor cells thatco-express NKX2.1GFP (top) and FOXG1 (bottom).

FIG. 24, D: B27+5F differentiation protocol with LDN193189 (0.1 μM,Catalog #04-0019 (Stemgent)) and IWP2 (5 μM, Stemgent Catalog #04-0034)as inhibitors of BMP and WNT signaling pathways, respectively, induceddifferentiation of hESCs into MGE precursor cells that co-expressNKX2.1GFP (top) and LHX6 (bottom).

FIG. 24, E: B27+5F differentiation protocol with Dorsamorphin (1 μM,Sigma Catalog # P5499) and (CKI)-7 (1 μM, Sigma Catalog #00742) asinhibitors of BMP and WNT signaling pathways, respectively, induceddifferentiation of hESCs into MGE precursor cells that co-expressNKX2.1GFP and NKX2.1.

The timing of addition of the inhibitors of BMP and WNT signalingpathways was as depicted in FIG. 1A.

FIG. 24, Bottom Panel: Flow cytometry was used to determine theefficiency of generation of NKX2.1GFP+MGE precursor cells using thesmall molecule inhibitors, The efficiency of generation of NKX2.1GFP+MGEprecursor cells was determined as what percent of cells of the cellsanalyzed were NKX2.1GFP+MGE precursor cells.

For the B27±5F differentiation protocol with BMPR1A and DKK1 asinhibitors of BMP and WNT signaling pathways, respectively, efficiencyof generation of was 81.6%. When LDN193189 and XAV939 were substitutedfor BMPR1A and DKK1, the efficiency of generation of NKX2.1GFP+MGE was86.9%. Substitution of BMPR1A and DKK1 with LDN193189 and IWR resultedin generation of NKX2.1GFP+ MGE at an efficiency of 89.3%. (FIG. 24,bottom panel). 30,000 to 100,000 cells were analyzed.

What is claimed is:
 1. A method of providing a cell culture enriched forGABAergic neuronal precursors, the method comprising: a) providingprimate pluripotent stem (pPS) cells in a culture medium; b) introducingto the culture medium, factors comprising: i) an activator of the sonichedgehog (shh) pathway, ii) a SMAD inhibitor, and iii) a wnt pathwayinhibitor, to produce a cell culture enriched in MGE precursor cells;and c) introducing a Notch pathway inhibitor to the cell cultureenriched in MGE precursor cells; wherein steps a) through c) result in acell culture enriched in GABAergic neuronal precursors.
 2. The method ofclaim 1, wherein the GABAergic neuronal precursors co-express NKX2.1-GFPand i12b-RFP.
 3. The method of claim 1, wherein the GABAergic neuronalprecursors have downregulated expression of NKX2.1.
 4. The method ofclaim 1, wherein the pPS cells are provided in a serum-free culturemedium.
 5. The method of claim 1, wherein the pPS cells are humaninduced pluripotent stem cells.
 6. The method of claim 1, wherein thepPS cells are human embryonic stem cells.
 7. The method of claim 1,wherein the pPS cells are cultured in the absence of a feeder layer. 8.The method of claim 1, wherein the pPS cells are cultured in suspension.9. The method of claim 1, wherein two or more inhibitors of SMAD areintroduced to the cells cultured to enrich for MGE precursor cells. 10.The method of claim 1, wherein an apoptosis inhibitor is introduced tothe cells cultured to enrich for MGE precursor cells.
 11. The method ofclaim 10, wherein the apoptosis inhibitor is an inhibitor ofRho-associated kinase (ROCK).
 12. The method of claim 1, wherein aneural supplement is added to the culture.
 13. The method of claim 1,wherein an anti-mitotic compound is added to the cell culture enrichedin MGE precursor cells to enrich for GABAergic neuronal precursors. 14.The method of claim 1, wherein a neurotrophic factor is added to theculture to enrich for GABAergic neuronal precursors.
 15. The method ofclaim 1, further comprising adding a cryoprotectant to the cell cultureenriched in GAB Aergic neuronal precursors.
 16. The method of claim 1,wherein the pPS cells are genetically modified.
 17. The method of claim16, wherein the pPS cells express a fluorescent protein.
 18. The methodof claim 1, further comprising isolating the GABAergic neuronalprecursors.
 19. The method of claim 18, wherein the isolating comprisesusing mechanical means.
 20. The method of claim 18, wherein theisolating comprises using an affinity reagent that binds to theGABAergic neuronal precursors.
 21. The method of claim 18, wherein theisolating comprises using enzymatic means.